Substituted Benzene Compounds

ABSTRACT

The present invention relates to substituted benzene compounds. The present invention also relates to pharmaceutical compositions containing these compounds and methods of treating cancer by administering these compounds and pharmaceutical compositions to subjects in need thereof. The present invention also relates to the use of such compounds for research or other non-therapeutic purposes.

RELATED APPLICATIONS

This application claims priority to, and the benefit of, U.S.provisional application Nos. 61/714,140, filed Oct. 15, 2012,61/714,145, filed Oct. 15, 2012, 61/780,703, filed Mar. 13, 2013, and61/786,277, filed Mar. 14, 2013. The entire contents of each of theseprovisional applications are incorporated herein by reference in theirentireties.

BACKGROUND OF THE INVENTION

There is an ongoing need for new agents as inhibitors of EZH2 activity,which can be used for treating EZH2-mediated disorder (e.g., cancer).

SUMMARY OF THE INVENTION

In one aspect, the present invention features a substituted benzenecompound selected from

and pharmaceutically acceptable salts thereof.

For example, the compound is

or a pharmaceutically acceptable salt thereof.

For example, the compound is

For example, the compound is

or a pharmaceutically acceptable salt thereof.

For example, the compound is

For example, the compound is

or a pharmaceutically acceptable salt thereof.

For example, the compound is

The present invention also provides pharmaceutical compositionscomprising one or more pharmaceutically acceptable carriers and one ormore compounds disclosed herein.

Another aspect of this invention is a method of treating or preventingan EZH2-mediated disorder. The method includes administering to asubject in need thereof a therapeutically effective amount of one ormore compounds disclosed herein. The EZH2-mediated disorder is adisease, disorder, or condition that is mediated at least in part by theactivity of EZH2. In one embodiment, the EZH2-mediated disorder isrelated to an increased EZH2 activity. In one embodiment, theEZH2-mediated disorder is a cancer. The EZH2-mediated cancer may belymphoma, leukemia or melanoma, for example, diffuse large B-celllymphoma (DLBCL), non-Hodgkin's lymphoma (NHL), follicular lymphoma,chronic myelogenous leukemia (CML), acute myeloid leukemia, acutelymphocytic leukemia, mixed lineage leukemia, or myelodysplasticsyndromes (MDS). In one embodiment the EZH2-mediated cancer may be amalignant rhabdoid tumor or INI1-defecient tumor. The histologicdiagnosis of malignant rhabdoid tumor depends on identification ofcharacteristic rhabdoid cells (large cells with eccentrically locatednuclei and abundant, eosinophilic cytoplasm) and immunohistochemistrywith antibodies to vimentin, keratin and epithelial membrane antigen. Inmost malignant rhabdoid tumors, the SMARCB1/INI1 gene, located inchromosome band 22q11.2, is inactivated by deletions and/or mutations.In one embodiment, the malignant rhabdoid tumors may be INI1-defecienttumor.

Unless otherwise stated, any description of a method of treatmentincludes uses of the compounds to provide such treatment or prophylaxisas is described in the specification, as well as uses of the compoundsto prepare a medicament to treat or prevent such condition. Thetreatment includes treatment of human or non-human animals includingrodents and other disease models.

Further, the compounds or methods described herein may be used forresearch (e.g., studying epigenetic enzymes) and other non-therapeuticpurposes.

In certain embodiments, the preferred compounds disclosed herein havedesirable pharmacological and/or pharmacokinetic properties, e.g., lowclearance rates and/or limited risk of adverse drug-drug interactions incombination therapy evaluated, for example, through time-dependent andreversible inhibition of cytochrome P-450 enzymes.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. In the specification, thesingular forms also include the plural unless the context clearlydictates otherwise. Although methods and materials similar or equivalentto those described herein can be used in the practice or testing of thepresent invention, suitable methods and materials are described below.All publications, patent applications, patents and other referencesmentioned herein are incorporated by reference. The references citedherein are not admitted to be prior art to the claimed invention. In thecase of conflict, the present specification, including definitions, willcontrol. In addition, the materials, methods and examples areillustrative only and are not intended to be limiting. In the case ofconflict between the chemical structures and names of the compoundsdisclosed herein, the chemical structures will control.

Other features and advantages of the invention will be apparent from thefollowing detailed description and claims.

BRIEF DESCRIPTIONS OF FIGURES

FIG. 1 is a diagram showing antitumor effects of orally administeredCompound 1 or Compound A against a lymphoma KARPAS-422 xenograft inmice. Data represent the mean±SD (n=10).

FIG. 2 is a diagram showing effect of Compound 1 or Compound A on mousebody weight. Data represent the mean±SD (n=10).

FIG. 3 is a diagram showing concentration of Compound 1 in tumor at day7 or day 28 post treatment or concentration of Compound A in tumor atday 7 post treatment. In this figure, “A” though “G” denote 7 days postadministration of Compound 1 at dosages of 62.5, 83.3, 125, 166.7, 250,333.3, and 500 mg/kg, respectively; “H” and “I” denote 7 days postadministration of Compound A at dosages of 125 and 250 mg/kg,respectively; and “J” through “L” denote 28 days post administration ofCompound 1 at dosages of 62.5, 125 and 250 mg/kg, respectively.

FIG. 4 is a diagram showing concentration of Compound 1 or Compound A inplasma at day 7 or day 28 post treatment. The top dashed line indicatesthe plasma protein binding (PPB) corrected LCC of Compound A and thebottom dashed line indicates PPB corrected LCC of Compound 1.

FIG. 5 is a diagram showing global H3K27me3 methylation in KARPAS-422tumors from mice treated with Compound 1 or Compound A for 7 days. Inthis figure, “A” denotes vehicle treatment; “B” though “H” denotetreatment with Compound 1 at dosages of 62.5, 83.3, 125, 166.7, 250,333.3, and 500 mg/kg, respectively; and “I” and “J” denote treatmentwith Compound A at dosages of 125 and 250 mg/kg, respectively.

FIG. 6 is a diagram showing global H3K27me3 methylation in KARPAS-422tumors from mice treated with Compound 1 for 28 days.

FIG. 7 is a diagram showing global H3K27me3 methylation in bone marrowfrom KARPAS-422 xenograft tumor bearing mice treated with Compound 1 orCompound A for 7 days. In this figure, “A” denotes vehicle treatment;“B” though “H” denote treatment with Compound 1 at dosages of 62.5,83.3, 125, 166.7, 250, 333.3, and 500 mg/kg, respectively; and “I” and“J” denote treatment with Compound A at dosages of 125 and 250 mg/kg,respectively.

FIG. 8 is a diagram showing global H3K27me3 methylation in bone marrowfrom KARPAS-422 xenograft tumor bearing mice treated with Compound 1 for28 days. In this figure, “A” denotes vehicle treatment; “B” though “E”denote treatment with Compound 1 at dosages of 62.5, 125, 250, and 500mg/kg, respectively; and “F” denotes treatment with Compound A at adosage of 250 mg/kg.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides novel substituted benzene compounds,synthetic methods for making the compounds, pharmaceutical compositionscontaining them and various uses of the compounds.

Representative compounds of the present invention include compoundslisted in Table 1.

TABLE 1 Com- pound no. Structure 1

2

105

In the present specification, the structural formula of the compoundrepresents a certain isomer for convenience in some cases, but thepresent invention may include all isomers, such as geometrical isomers,optical isomers based on an asymmetrical carbon, stereoisomers,tautomers, enantiomers, rotamers, diastereomers, racemates and the like,it being understood that not all isomers may have the same level ofactivity. In addition, a crystal polymorphism may be present for thecompounds represented by the formula. It is noted that any crystal form,crystal form mixture, or anhydride or hydrate thereof is included in thescope of the present invention.

“Isomerism” means compounds that have identical molecular formulae butdiffer in the sequence of bonding of their atoms or in the arrangementof their atoms in space. Isomers that differ in the arrangement of theiratoms in space are termed “stereoisomers.” Stereoisomers that are notmirror images of one another are termed “diastereoisomers,” andstereoisomers that are non-superimposable mirror images of each otherare termed “enantiomers” or sometimes optical isomers. A mixturecontaining equal amounts of individual enantiomeric forms of oppositechirality is termed a “racemic mixture.”

“Geometric isomer” means the diastereomers that owe their existence tohindered rotation about double bonds or a cycloalkyl linker (e.g.,1,3-cylcobutyl). These configurations are differentiated in their namesby the prefixes cis and trans, or Z and E, which indicate that thegroups are on the same or opposite side of the double bond in themolecule according to the Cahn-Ingold-Prelog rules.

It is to be understood that the compounds of the present invention maybe depicted as stereoisomers. It should also be understood that whencompounds have stereoisomeric forms, all stereoisomeric forms areintended to be included in the scope of the present invention, it beingunderstood that not all stereoisomers may have the same level ofactivity.

Furthermore, the structures and other compounds discussed in thisinvention include all atropic isomers thereof, it being understood thatnot all atropic isomers may have the same level of activity. “Atropicisomers” are a type of stereoisomer in which the atoms of two isomersare arranged differently in space. Atropic isomers owe their existenceto a restricted rotation caused by hindrance of rotation of large groupsabout a central bond. Such atropic isomers typically exist as a mixture,however as a result of recent advances in chromatography techniques, ithas been possible to separate mixtures of two atropic isomers in selectcases.

“Tautomer” is one of two or more structural isomers that exist inequilibrium and is readily converted from one isomeric form to another.This conversion results in the formal migration of a hydrogen atomaccompanied by a switch of adjacent conjugated double bonds. Tautomersexist as a mixture of a tautomeric set in solution. In solutions wheretautomerization is possible, a chemical equilibrium of the tautomerswill be reached. The exact ratio of the tautomers depends on severalfactors, including temperature, solvent and pH. The concept of tautomersthat are interconvertable by tautomerizations is called tautomerism.

Of the various types of tautomerism that are possible, two are commonlyobserved. In keto-enol tautomerism a simultaneous shift of electrons anda hydrogen atom occurs. Ring-chain tautomerism arises as a result of thealdehyde group (—CHO) in a sugar chain molecule reacting with one of thehydroxy groups (—OH) in the same molecule to give it a cyclic(ring-shaped) form as exhibited by glucose.

As used herein, any occurrence of

should be construed as

Common tautomeric pairs are: ketone-enol, amide-nitrile, lactam-lactim,amide-imidic acid tautomerism in heterocyclic rings (e.g., innucleobases such as guanine, thymine and cytosine), imine-enamine andenamine-enamine. An example of keto-enol equilibria is betweenpyridin-2(1H)-ones and the corresponding pyridin-2-ols, as shown below.

It is to be understood that the compounds of the present invention maybe depicted as different tautomers. It should also be understood thatwhen compounds have tautomeric forms, all tautomeric forms are intendedto be included in the scope of the present invention, and the naming ofthe compounds does not exclude any tautomer form. It will be understoodthat certain tautomers may have a higher level of activity than others.

The term “crystal polymorphs”, “polymorphs” or “crystal forms” meanscrystal structures in which a compound (or a salt or solvate thereof)can crystallize in different crystal packing arrangements, all of whichhave the same elemental composition. Different crystal forms usuallyhave different X-ray diffraction patterns, infrared spectral, meltingpoints, density hardness, crystal shape, optical and electricalproperties, stability and solubility. Recrystallization solvent, rate ofcrystallization, storage temperature, and other factors may cause onecrystal form to dominate. Crystal polymorphs of the compounds can beprepared by crystallization under different conditions.

The compounds of this invention include the compounds themselves, suchas any of the formulae disclosed herein. The compounds of this inventionmay also include their salts, and their solvates, if applicable. A salt,for example, can be formed between an anion and a positively chargedgroup (e.g., amino) on a substituted benzene compound. Suitable anionsinclude chloride, bromide, iodide, sulfate, bisulfate, sulfamate,nitrate, phosphate, citrate, methanesulfonate, trifluoroacetate,glutamate, glucuronate, glutarate, malate, maleate, succinate, fumarate,tartrate, tosylate, salicylate, lactate, naphthalenesulfonate, andacetate (e.g., trifluoroacetate). The term “pharmaceutically acceptableanion” refers to an anion suitable for forming a pharmaceuticallyacceptable salt. Likewise, a salt can also be formed between a cationand a negatively charged group (e.g., carboxylate) on a substitutedbenzene compound. Suitable cations include sodium ion, potassium ion,magnesium ion, calcium ion, and an ammonium cation such astetramethylammonium ion. The substituted benzene compounds also includethose salts containing quaternary nitrogen atoms.

Additionally, the compounds of the present invention, for example, thesalts of the compounds, can exist in either hydrated or unhydrated (theanhydrous) form or as solvates with other solvent molecules. Nonlimitingexamples of hydrates include monohydrates, dihydrates, etc. Nonlimitingexamples of solvates include ethanol solvates, acetone solvates, etc.

“Solvate” means solvent addition forms that contain eitherstoichiometric or non stoichiometric amounts of solvent. Some compoundshave a tendency to trap a fixed molar ratio of solvent molecules in thecrystalline solid state, thus forming a solvate. If the solvent is waterthe solvate formed is a hydrate; and if the solvent is alcohol, thesolvate formed is an alcoholate. Hydrates are formed by the combinationof one or more molecules of water with one molecule of the substance inwhich the water retains its molecular state as H₂O.

As used herein, the term “analog” refers to a chemical compound that isstructurally similar to another but differs slightly in composition (asin the replacement of one atom by an atom of a different element or inthe presence of a particular functional group, or the replacement of onefunctional group by another functional group). Thus, an analog is acompound that is similar or comparable in function and appearance, butnot in structure or origin to the reference compound.

As defined herein, the term “derivative” refers to compounds that have acommon core structure, and are substituted with various groups asdescribed herein. For example, all of the compounds in Table 1 aresubstituted benzene compounds, and have a common core.

The term “bioisostere” refers to a compound resulting from the exchangeof an atom or of a group of atoms with another, broadly similar, atom orgroup of atoms. The objective of a bioisosteric replacement is to createa new compound with similar biological properties to the parentcompound. The bioisosteric replacement may be physicochemically ortopologically based. Examples of carboxylic acid bioisosteres include,but are not limited to, acyl sulfonimides, tetrazoles, sulfonates andphosphonates. See, e.g., Patani and LaVoie, Chem. Rev. 96, 3147-3176,1996.

The present invention is intended to include all isotopes of atomsoccurring in the present compounds. Isotopes include those atoms havingthe same atomic number but different mass numbers. By way of generalexample and without limitation, isotopes of hydrogen include tritium anddeuterium, and isotopes of carbon include C-13 and C-14.

The present invention provides methods for the synthesis of thecompounds disclosed herein. The present invention also provides detailedmethods for the synthesis of various disclosed compounds of the presentinvention according to the schemes as shown in the Examples.

Throughout the description, where compositions are described as having,including, or comprising specific components, it is contemplated thatcompositions also consist essentially of or consist of the recitedcomponents. Similarly, where methods or processes are described ashaving, including, or comprising specific process steps, the processesalso consist essentially of, or consist of, the recited processingsteps. Further, it should be understood that the order of steps or orderfor performing certain actions is immaterial so long as the inventionremains operable. Moreover, two or more steps or actions can beconducted simultaneously.

The synthetic processes of the invention can tolerate a wide variety offunctional groups, therefore various substituted starting materials canbe used. The processes generally provide the desired final compound ator near the end of the overall process, although it may be desirable incertain instances to further convert the compound to a pharmaceuticallyacceptable salt thereof.

Compounds of the present invention can be prepared in a variety of waysusing commercially available starting materials, compounds known in theliterature, or from readily prepared intermediates, by employingstandard synthetic methods and procedures either known to those skilledin the art, or which will be apparent to the skilled artisan in light ofthe teachings herein. Standard synthetic methods and procedures for thepreparation of organic molecules and functional group transformationsand manipulations can be obtained from the relevant scientificliterature or from standard textbooks in the field. Although not limitedto any one or several sources, classic texts such as Smith, M. B.,March, J., March's Advanced Organic Chemistry: Reactions, Mechanisms,and Structure, 5^(th) edition, John Wiley & Sons: New York, 2001;Greene, T. W., Wuts, P. G. M., Protective Groups in Organic Synthesis,3^(rd) edition, John Wiley & Sons: New York, 1999; R. Larock,Comprehensive Organic Transformations, VCH Publishers (1989); L. Fieserand M. Fieser, Fieser and Fieser's Reagents for Organic Synthesis, JohnWiley and Sons (1994); and L. Paquette, ed., Encyclopedia of Reagentsfor Organic Synthesis, John Wiley and Sons (1995), incorporated byreference herein, are useful and recognized reference textbooks oforganic synthesis known to those in the art. The following descriptionsof synthetic methods are designed to illustrate, but not to limit,general procedures for the preparation of compounds of the presentinvention.

One of ordinary skill in the art will recognize that certain groups mayrequire protection from the reaction conditions via the use ofprotecting groups. Protecting groups may also be used to differentiatesimilar functional groups in molecules. A list of protecting groups andhow to introduce and remove these groups can be found in Greene, T. W.,Wuts, P. G. M., Protective Groups in Organic Synthesis, 3^(rd) edition,John Wiley & Sons: New York, 1999.

Preferred protecting groups include, but are not limited to:

For a hydroxyl moiety: TBS, benzyl, THP, Ac

For carboxylic acids: benzyl ester, methyl ester, ethyl ester, allylester

For amines: Cbz, BOC, DMB

For diols: Ac (x2) TBS (x2), or when taken together acetonides

For thiols: Ac

For benzimidazoles: SEM, benzyl, PMB, DMB

For aldehydes: di-alkyl acetals such as dimethoxy acetal or diethylacetyl.

In the reaction schemes described herein, multiple stereoisomers may beproduced. When no particular stereoisomer is indicated, it is understoodto mean all possible stereoisomers that could be produced from thereaction. A person of ordinary skill in the art will recognize that thereactions can be optimized to give one isomer preferentially, or newschemes may be devised to produce a single isomer. If mixtures areproduced, techniques such as preparative thin layer chromatography,preparative HPLC, preparative chiral HPLC, or preparative SFC may beused to separate the isomers.

The following abbreviations are used throughout the specification andare defined below:

Ac acetyl

AcOH acetic acid

aq. aqueous

BID or b.i.d. bis in die (twice a day)

BOC tert-butoxy carbonyl

Cbz benzyloxy carbonyl

CDCl₃ deuterated chloroform

CH₂Cl₂ dichloromethane

DCM dichloromethane

DMB 2,4 dimethoxy benzyl

DMF N,N-Dimethylformamide

DMSO Dimethyl sulfoxide

EA or EtOAc Ethyl acetate

EDC or EDCI N-(3-Dimethylaminopropyl)-N′-ethylcarbodiimide

ESI− Electrospray negative mode

ESI+ Electrospray positive mode

EtOH ethanol

h hours

H₂O water

HOBt 1-Hydroxybenzotriazole

HCl hydrogen chloride or hydrochloric acid

HPLC High performance liquid chromatography

K₂CO₃ potassium carbonate

LC/MS or LC-MS Liquid chromatography mass spectrum

M Molar

MeCN Acetonitrile

min minutes

Na₂CO₃ sodium carbonate

Na₂SO₄ sodium sulfate

NaHCO₃ sodium bicarbonate

NaHMDs Sodium hexamethyldisilazide

NaOH sodium hydroxide

NaHCO₃ sodium bicarbonate

Na₂SO₄ sodium sulfate

NMR Nuclear Magnetic Resonance

Pd(OH)₂ Palladium dihydroxide

PMB para methoxybenzyl

p.o. per os (oral administration)

ppm parts per million

prep HPLC preparative High Performance Liquid Chromatography

PYBOP (Benzotriazol-1-yloxy)tripyrrolidinophosphoniumhexafluorophosphate

Rt or RT Room temperature

TBME tert-Butyl methyl ether

TFA trifluoroacetic acid

THF tetrahydrofuran

THP tetrahydropyran

Compounds of the present invention can be conveniently prepared by avariety of methods familiar to those skilled in the art. The compoundsof this invention with any Formula disclosed herein may be preparedaccording to the procedures illustrated in the Examples below, fromcommercially available starting materials or starting materials whichcan be prepared using literature procedures.

One of ordinary skill in the art will note that, during the reactionsequences and synthetic schemes described herein, the order of certainsteps may be changed, such as the introduction and removal of protectinggroups.

Compounds of the present invention inhibit the histone methyltransferaseactivity of EZH2 or a mutant thereof and, accordingly, in one aspect ofthe invention, certain compounds disclosed herein are candidates fortreating, or preventing certain conditions and diseases in which EZH2plays a role. The present invention provides methods for treatingconditions and diseases the course of which can be influenced bymodulating the methylation status of histones or other proteins, whereinsaid methylation status is mediated at least in part by the activity ofEZH2. Modulation of the methylation status of histones can in turninfluence the level of expression of target genes activated bymethylation, and/or target genes suppressed by methylation. The methodincludes administering to a subject in need of such treatment, atherapeutically effective amount of a compound of the present invention,or a pharmaceutically acceptable salt, polymorph, solvate, orstereoisomeror thereof.

Unless otherwise stated, any description of a method of treatmentincludes uses of the compounds to provide such treatment or prophylaxisas is described in the specification, as well as uses of the compoundsto prepare a medicament to treat or prevent such condition. Thetreatment includes treatment of human or non-human animals includingrodents and other disease models.

In still another aspect, this invention relates to a method ofmodulating the activity of the EZH2, the catalytic subunit of the PRC2complex which catalyzes the mono- through tri-methylation of lysine 27on histone H3 (H3-K27) in a subject in need thereof. For example, themethod comprises the step of administering to a subject having a cancerexpressing a mutant EZH2 a therapeutically effective amount of acompound described herein, wherein the compound(s) inhibits histonemethyltransferase activity of EZH2, thereby treating the cancer.

For example, the EZH2-mediated cancer is selected from the groupconsisting of follicular lymphoma and diffuse large B-cell lymphoma(DLBCL) of germinal center B cell-like (GCB) subtype. For example, thecancer is lymphoma, leukemia or melanoma. Preferably, the lymphoma isnon-Hodgkin's lymphoma (NHL), follicular lymphoma or diffuse largeB-cell lymphoma. Alternatively, the leukemia is chronic myelogenousleukemia (CML), acute myeloid leukemia, acute lymphocytic leukemia ormixed lineage leukemia.

For example, the EZH2-mediated precancerous condition is myelodysplasticsyndromes (MDS, formerly known as preleukemia).

For example, the EZH2-mediated cancer is a hematological cancer.

The compound(s) of the present invention inhibit the histonemethyltransferase activity of EZH2 or a mutant thereof and, accordingly,the present invention also provides methods for treating conditions anddiseases the course of which can be influenced by modulating themethylation status of histones or other proteins, wherein saidmethylation status is mediated at least in part by the activity of EZH2.In one aspect of the invention, certain compounds disclosed herein arecandidates for treating, or preventing certain conditions and diseases.Modulation of the methylation status of histones can in turn influencethe level of expression of target genes activated by methylation, and/ortarget genes suppressed by methylation. The method includesadministering to a subject in need of such treatment, a therapeuticallyeffective amount of a compound of the present invention.

As used herein, a “subject” is interchangeable with a “subject in needthereof”, both of which refer to a subject having a disorder in whichEZH2-mediated protein methylation plays a part, or a subject having anincreased risk of developing such disorder relative to the population atlarge. A “subject” includes a mammal. The mammal can be e.g., a human orappropriate non-human mammal, such as primate, mouse, rat, dog, cat,cow, horse, goat, camel, sheep or a pig. The subject can also be a birdor fowl. In one embodiment, the mammal is a human. A subject in needthereof can be one who has been previously diagnosed or identified ashaving cancer or a precancerous condition. A subject in need thereof canalso be one who has (e.g., is suffering from) cancer or a precancerouscondition. Alternatively, a subject in need thereof can be one who hasan increased risk of developing such disorder relative to the populationat large (i.e., a subject who is predisposed to developing such disorderrelative to the population at large). A subject in need thereof can havea precancerous condition. A subject in need thereof can have refractoryor resistant cancer (i.e., cancer that doesn't respond or hasn't yetresponded to treatment). The subject may be resistant at start oftreatment or may become resistant during treatment. In some embodiments,the subject in need thereof has cancer recurrence following remission onmost recent therapy. In some embodiments, the subject in need thereofreceived and failed all known effective therapies for cancer treatment.In some embodiments, the subject in need thereof received at least oneprior therapy. In a preferred embodiment, the subject has cancer or acancerous condition. For example, the cancer is lymphoma, leukemia,melanoma, or rhabdomyosarcoma. Preferably, the lymphoma is non-Hodgkin'slymphoma, follicular lymphoma or diffuse large B-cell lymphoma.Alternatively, the leukemia is chronic myelogenous leukemia (CML). Theprecancerous condition is myelodysplastic syndromes (MDS, formerly knownas preleukemia).

As used herein, “treating” or “treat” describes the management and careof a patient for the purpose of combating a disease, condition, ordisorder and includes the administration of a compound of the presentinvention, or a pharmaceutically acceptable salt, polymorph or solvatethereof, to alleviate the symptoms or complications of a disease,condition or disorder, or to eliminate the disease, condition ordisorder. The term “treat” can also include treatment of a cell in vitroor an animal model.

A compound of the present invention, or a pharmaceutically acceptablesalt, polymorph or solvate thereof, can or may also be used to prevent arelevant disease, condition or disorder, or used to identify suitablecandidates for such purposes. As used herein, “preventing,” “prevent,”or “protecting against” describes reducing or eliminating the onset ofthe symptoms or complications of such disease, condition or disorder.

Point mutations of the EZH2 gene at a single amino acid residue (e.g.,Y641, A677, and A687) of EZH2 have been reported to be linked tolymphoma. More examples of EZH2 mutants and methods of detection ofmutation and methods treatment of mutation-associated disorders aredescribed in, e.g., U.S. Patent Application Publication No. US20130040906, the entire content of which is incorporated herein byreference in its entirety.

One skilled in the art may refer to general reference texts for detaileddescriptions of known techniques discussed herein or equivalenttechniques. These texts include Ausubel et al., Current Protocols inMolecular Biology, John Wiley and Sons, Inc. (2005); Sambrook et al.,Molecular Cloning, A Laboratory Manual (3^(rd) edition), Cold SpringHarbor Press, Cold Spring Harbor, N.Y. (2000); Coligan et al., CurrentProtocols in Immunology, John Wiley & Sons, N.Y.; Enna et al., CurrentProtocols in Pharmacology, John Wiley & Sons, N.Y.; Fingl et al., ThePharmacological Basis of Therapeutics (1975), Remington's PharmaceuticalSciences, Mack Publishing Co., Easton, Pa., 18^(th) edition (1990).These texts can, of course, also be referred to in making or using anaspect of the invention.

As used herein, “combination therapy” or “co-therapy” includes theadministration of a compound of the present invention, or apharmaceutically acceptable salt, polymorph or solvate thereof, and atleast a second agent as part of a specific treatment regimen intended toprovide the beneficial effect from the co-action of these therapeuticagents. The beneficial effect of the combination includes, but is notlimited to, pharmacokinetic or pharmacodynamic co-action resulting fromthe combination of therapeutic agents.

The present invention also provides pharmaceutical compositionscomprising a compound disclosed herein in combination with at least onepharmaceutically acceptable excipient or carrier.

A “pharmaceutical composition” is a formulation containing the compoundsof the present invention in a form suitable for administration to asubject. In one embodiment, the pharmaceutical composition is in bulk orin unit dosage form. The unit dosage form is any of a variety of forms,including, for example, a capsule, an IV bag, a tablet, a single pump onan aerosol inhaler or a vial. The quantity of active ingredient (e.g., aformulation of the disclosed compound or salt, hydrate, solvate orisomer thereof) in a unit dose of composition is an effective amount andis varied according to the particular treatment involved. One skilled inthe art will appreciate that it is sometimes necessary to make routinevariations to the dosage depending on the age and condition of thepatient. The dosage will also depend on the route of administration. Avariety of routes are contemplated, including oral, pulmonary, rectal,parenteral, transdermal, subcutaneous, intravenous, intramuscular,intraperitoneal, inhalational, buccal, sublingual, intrapleural,intrathecal, intranasal, and the like. Dosage forms for the topical ortransdermal administration of a compound of this invention includepowders, sprays, ointments, pastes, creams, lotions, gels, solutions,patches and inhalants. In one embodiment, the active compound is mixedunder sterile conditions with a pharmaceutically acceptable carrier, andwith any preservatives, buffers, or propellants that are required.

As used herein, the phrase “pharmaceutically acceptable” refers to thosecompounds, anions, cations, materials, compositions, carriers, and/ordosage forms which are, within the scope of sound medical judgment,suitable for use in contact with the tissues of human beings and animalswithout excessive toxicity, irritation, allergic response, or otherproblem or complication, commensurate with a reasonable benefit/riskratio.

“Pharmaceutically acceptable excipient” means an excipient that isuseful in preparing a pharmaceutical composition that is generally safe,non-toxic and neither biologically nor otherwise undesirable, andincludes excipient that is acceptable for veterinary use as well ashuman pharmaceutical use. A “pharmaceutically acceptable excipient” asused in the specification and claims includes both one and more than onesuch excipient.

A pharmaceutical composition of the invention is formulated to becompatible with its intended route of administration. Examples of routesof administration include parenteral, e.g., intravenous, intradermal,subcutaneous, oral (e.g., inhalation), transdermal (topical), andtransmucosal administration. Solutions or suspensions used forparenteral, intradermal, or subcutaneous application can include thefollowing components: a sterile diluent such as water for injection,saline solution, fixed oils, polyethylene glycols, glycerine, propyleneglycol or other synthetic solvents; antibacterial agents such as benzylalcohol or methyl parabens; antioxidants such as ascorbic acid or sodiumbisulfate; chelating agents such as ethylenediaminetetraacetic acid;buffers such as acetates, citrates or phosphates, and agents for theadjustment of tonicity such as sodium chloride or dextrose. The pH canbe adjusted with acids or bases, such as hydrochloric acid or sodiumhydroxide. The parenteral preparation can be enclosed in ampoules,disposable syringes or multiple dose vials made of glass or plastic.

A compound or pharmaceutical composition of the invention can beadministered to a subject in many of the well-known methods currentlyused for chemotherapeutic treatment. For example, for treatment ofcancers, a compound of the invention may be injected directly intotumors, injected into the blood stream or body cavities or taken orallyor applied through the skin with patches. The dose chosen should besufficient to constitute effective treatment but not so high as to causeunacceptable side effects. The state of the disease condition (e.g.,cancer, precancer, and the like) and the health of the patient shouldpreferably be closely monitored during and for a reasonable period aftertreatment.

The term “therapeutically effective amount”, as used herein, refers toan amount of a pharmaceutical agent to treat, ameliorate, or prevent anidentified disease or condition, or to exhibit a detectable therapeuticor inhibitory effect. The effect can be detected by any assay methodknown in the art. The precise effective amount for a subject will dependupon the subject's body weight, size, and health; the nature and extentof the condition; and the therapeutic or combination of therapeuticsselected for administration. Therapeutically effective amounts for agiven situation can be determined by routine experimentation that iswithin the skill and judgment of the clinician. In a preferred aspect,the disease or condition to be treated is cancer. In another aspect, thedisease or condition to be treated is a cell proliferative disorder.

For any compound, the therapeutically effective amount can be estimatedinitially either in cell culture assays, e.g., of neoplastic cells, orin animal models, usually rats, mice, rabbits, dogs, or pigs. The animalmodel may also be used to determine the appropriate concentration rangeand route of administration. Such information can then be used todetermine useful doses and routes for administration in humans.Therapeutic/prophylactic efficacy and toxicity may be determined bystandard pharmaceutical procedures in cell cultures or experimentalanimals, e.g., ED₅₀ (the dose therapeutically effective in 50% of thepopulation) and LD₅₀ (the dose lethal to 50% of the population). Thedose ratio between toxic and therapeutic effects is the therapeuticindex, and it can be expressed as the ratio, LD₅₀/ED₅₀. Pharmaceuticalcompositions that exhibit large therapeutic indices are preferred. Thedosage may vary within this range depending upon the dosage formemployed, sensitivity of the patient, and the route of administration.

Dosage and administration are adjusted to provide sufficient levels ofthe active agent(s) or to maintain the desired effect. Factors which maybe taken into account include the severity of the disease state, generalhealth of the subject, age, weight, and gender of the subject, diet,time and frequency of administration, drug combination(s), reactionsensitivities, and tolerance/response to therapy. Long-actingpharmaceutical compositions may be administered every 3 to 4 days, everyweek, or once every two weeks depending on half-life and clearance rateof the particular formulation.

The pharmaceutical compositions containing active compounds of thepresent invention may be manufactured in a manner that is generallyknown, e.g., by means of conventional mixing, dissolving, granulating,dragee-making, levigating, emulsifying, encapsulating, entrapping, orlyophilizing processes. Pharmaceutical compositions may be formulated ina conventional manner using one or more pharmaceutically acceptablecarriers comprising excipients and/or auxiliaries that facilitateprocessing of the active compounds into preparations that can be usedpharmaceutically. Of course, the appropriate formulation is dependentupon the route of administration chosen.

Pharmaceutical compositions suitable for injectable use include sterileaqueous solutions (where water soluble) or dispersions and sterilepowders for the extemporaneous preparation of sterile injectablesolutions or dispersion. For intravenous administration, suitablecarriers include physiological saline, bacteriostatic water, CremophorEL™ (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In allcases, the composition must be sterile and should be fluid to the extentthat easy syringeability exists. It must be stable under the conditionsof manufacture and storage and must be preserved against thecontaminating action of microorganisms such as bacteria and fungi. Thecarrier can be a solvent or dispersion medium containing, for example,water, ethanol, polyol (for example, glycerol, propylene glycol, andliquid polyethylene glycol, and the like), and suitable mixturesthereof. The proper fluidity can be maintained, for example, by the useof a coating such as lecithin, by the maintenance of the requiredparticle size in the case of dispersion and by the use of surfactants.Prevention of the action of microorganisms can be achieved by variousantibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In manycases, it will be preferable to include isotonic agents, for example,sugars, polyalcohols such as manitol and sorbitol, and sodium chloridein the composition. Prolonged absorption of the injectable compositionscan be brought about by including in the composition an agent whichdelays absorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions can be prepared by incorporating the activecompound in the required amount in an appropriate solvent with one or acombination of ingredients enumerated above, as required, followed byfiltered sterilization. Generally, dispersions are prepared byincorporating the active compound into a sterile vehicle that contains abasic dispersion medium and the required other ingredients from thoseenumerated above. In the case of sterile powders for the preparation ofsterile injectable solutions, methods of preparation are vacuum dryingand freeze-drying that yields a powder of the active ingredient plus anyadditional desired ingredient from a previously sterile-filteredsolution thereof.

Oral compositions generally include an inert diluent or an ediblepharmaceutically acceptable carrier. They can be enclosed in gelatincapsules or compressed into tablets. For the purpose of oral therapeuticadministration, the active compound can be incorporated with excipientsand used in the form of tablets, troches, or capsules. Oral compositionscan also be prepared using a fluid carrier for use as a mouthwash,wherein the compound in the fluid carrier is applied orally and swishedand expectorated or swallowed. Pharmaceutically compatible bindingagents, and/or adjuvant materials can be included as part of thecomposition. The tablets, pills, capsules, troches and the like cancontain any of the following ingredients, or compounds of a similarnature: a binder such as microcrystalline cellulose, gum tragacanth orgelatin; an excipient such as starch or lactose, a disintegrating agentsuch as alginic acid, Primogel, or corn starch; a lubricant such asmagnesium stearate or Sterotes; a glidant such as colloidal silicondioxide; a sweetening agent such as sucrose or saccharin; or a flavoringagent such as peppermint, methyl salicylate, or orange flavoring.

For administration by inhalation, the compounds are delivered in theform of an aerosol spray from pressured container or dispenser, whichcontains a suitable propellant, e.g., a gas such as carbon dioxide, or anebulizer.

Systemic administration can also be by transmucosal or transdermalmeans. For transmucosal or transdermal administration, penetrantsappropriate to the barrier to be permeated are used in the formulation.Such penetrants are generally known in the art, and include, forexample, for transmucosal administration, detergents, bile salts, andfusidic acid derivatives. Transmucosal administration can beaccomplished through the use of nasal sprays or suppositories. Fortransdermal administration, the active compounds are formulated intoointments, salves, gels, or creams as generally known in the art.

The active compounds can be prepared with pharmaceutically acceptablecarriers that will protect the compound against rapid elimination fromthe body, such as a controlled release formulation, including implantsand microencapsulated delivery systems. Biodegradable, biocompatiblepolymers can be used, such as ethylene vinyl acetate, polyanhydrides,polyglycolic acid, collagen, polyorthoesters, and polylactic acid.Methods for preparation of such formulations will be apparent to thoseskilled in the art. The materials can also be obtained commercially fromAlza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions(including liposomes targeted to infected cells with monoclonalantibodies to viral antigens) can also be used as pharmaceuticallyacceptable carriers. These can be prepared according to methods known tothose skilled in the art, for example, as described in U.S. Pat. No.4,522,811.

It is especially advantageous to formulate oral or parenteralcompositions in dosage unit form for ease of administration anduniformity of dosage. Dosage unit form as used herein refers tophysically discrete units suited as unitary dosages for the subject tobe treated; each unit containing a predetermined quantity of activecompound calculated to produce the desired therapeutic effect inassociation with the required pharmaceutical carrier. The specificationfor the dosage unit forms of the invention are dictated by and directlydependent on the unique characteristics of the active compound and theparticular therapeutic effect to be achieved.

In therapeutic applications, the dosages of the pharmaceuticalcompositions used in accordance with the invention vary depending on theagent, the age, weight, and clinical condition of the recipient patient,and the experience and judgment of the clinician or practitioneradministering the therapy, among other factors affecting the selecteddosage. Generally, the dose should be sufficient to result in slowing,and preferably regressing, the growth of the tumors and also preferablycausing complete regression of the cancer. Dosages can range from about0.01 mg/kg per day to about 5000 mg/kg per day. In preferred aspects,dosages can range from about 1 mg/kg per day to about 1000 mg/kg perday. In an aspect, the dose will be in the range of about 0.1 mg/day toabout 50 g/day; about 0.1 mg/day to about 25 g/day; about 0.1 mg/day toabout 10 g/day; about 0.1 mg to about 3 g/day; or about 0.1 mg to about1 g/day, in single, divided, or continuous doses (which dose may beadjusted for the patient's weight in kg, body surface area in m², andage in years). An effective amount of a pharmaceutical agent is thatwhich provides an objectively identifiable improvement as noted by theclinician or other qualified observer. For example, regression of atumor in a patient may be measured with reference to the diameter of atumor. Decrease in the diameter of a tumor indicates regression.Regression is also indicated by failure of tumors to reoccur aftertreatment has stopped. As used herein, the term “dosage effectivemanner” refers to amount of an active compound to produce the desiredbiological effect in a subject or cell.

The pharmaceutical compositions can be included in a container, pack, ordispenser together with instructions for administration.

The compounds of the present invention are capable of further formingsalts. All of these forms are also contemplated within the scope of theclaimed invention.

As used herein, “pharmaceutically acceptable salts” refer to derivativesof the compounds of the present invention wherein the parent compound ismodified by making acid or base salts thereof. Examples ofpharmaceutically acceptable salts include, but are not limited to,mineral or organic acid salts of basic residues such as amines, alkalior organic salts of acidic residues such as carboxylic acids, and thelike. The pharmaceutically acceptable salts include the conventionalnon-toxic salts or the quaternary ammonium salts of the parent compoundformed, for example, from non-toxic inorganic or organic acids. Forexample, such conventional non-toxic salts include, but are not limitedto, those derived from inorganic and organic acids selected from2-acetoxybenzoic, 2-hydroxyethane sulfonic, acetic, ascorbic, benzenesulfonic, benzoic, bicarbonic, carbonic, citric, edetic, ethanedisulfonic, 1,2-ethane sulfonic, fumaric, glucoheptonic, gluconic,glutamic, glycolic, glycollyarsanilic, hexylresorcinic, hydrabamic,hydrobromic, hydrochloric, hydroiodic, hydroxymaleic, hydroxynaphthoic,isethionic, lactic, lactobionic, lauryl sulfonic, maleic, malic,mandelic, methane sulfonic, napsylic, nitric, oxalic, pamoic,pantothenic, phenylacetic, phosphoric, polygalacturonic, propionic,salicyclic, stearic, subacetic, succinic, sulfamic, sulfanilic,sulfuric, tannic, tartaric, toluene sulfonic, and the commonly occurringamine acids, e.g., glycine, alanine, phenylalanine, arginine, etc.

Other examples of pharmaceutically acceptable salts include hexanoicacid, cyclopentane propionic acid, pyruvic acid, malonic acid,3-(4-hydroxybenzoyl)benzoic acid, cinnamic acid, 4-chlorobenzenesulfonicacid, 2-naphthalenesulfonic acid, 4-toluenesulfonic acid,camphorsulfonic acid, 4-methylbicyclo-[2.2.2]-oct-2-ene-1-carboxylicacid, 3-phenylpropionic acid, trimethylacetic acid, tertiary butylaceticacid, muconic acid, and the like. The present invention also encompassessalts formed when an acidic proton present in the parent compound eitheris replaced by a metal ion, e.g., an alkali metal ion, an alkaline earthion, or an aluminum ion; or coordinates with an organic base such asethanolamine, diethanolamine, triethanolamine, tromethamine,N-methylglucamine, and the like. In the salt form, it is understood thatthe ratio of the compound to the cation or anion of the salt can be 1:1,or any ration other than 1:1, e.g., 3:1, 2:1, 1:2, or 1:3.

It should be understood that all references to pharmaceuticallyacceptable salts include solvent addition forms (solvates) or crystalforms (polymorphs) as defined herein, of the same salt.

The compounds, or pharmaceutically acceptable salts thereof, areadministered orally, nasally, transdermally, pulmonary, inhalationally,buccally, sublingually, intraperintoneally, subcutaneously,intramuscularly, intravenously, rectally, intrapleurally, intrathecallyand parenterally. In one embodiment, the compound is administeredorally. One skilled in the art will recognize the advantages of certainroutes of administration.

The dosage regimen utilizing the compounds is selected in accordancewith a variety of factors including type, species, age, weight, sex andmedical condition of the patient; the severity of the condition to betreated; the route of administration; the renal and hepatic function ofthe patient; and the particular compound or salt thereof employed. Anordinarily skilled physician or veterinarian can readily determine andprescribe the effective amount of the drug required to prevent, counter,or arrest the progress of the condition.

Techniques for formulation and administration of the disclosed compoundsof the invention can be found in Remington: the Science and Practice ofPharmacy, 19^(th) edition, Mack Publishing Co., Easton, Pa. (1995). Inan embodiment, the compounds described herein, and the pharmaceuticallyacceptable salts thereof, are used in pharmaceutical preparations incombination with a pharmaceutically acceptable carrier or diluent.Suitable pharmaceutically acceptable carriers include inert solidfillers or diluents and sterile aqueous or organic solutions. Thecompounds will be present in such pharmaceutical compositions in amountssufficient to provide the desired dosage amount in the range describedherein.

All percentages and ratios used herein, unless otherwise indicated, areby weight. Other features and advantages of the present invention areapparent from the different examples. The provided examples illustratedifferent components and methodology useful in practicing the presentinvention. The examples do not limit the claimed invention. Based on thepresent disclosure the skilled artisan can identify and employ othercomponents and methodology useful for practicing the present invention.

In the synthetic schemes and chemical structures described herein,compounds may be drawn with one particular configuration (e.g., with orwithout a particular stereoisomer indicated) for simplicity. Suchparticular configurations or lack thereof are not to be construed aslimiting the invention to one or another isomer, tautomer, regioisomeror stereoisomer, nor does it exclude mixtures of isomers, tautomers,regioisomers or stereoisomers; however, it will be understood that agiven isomer, tautomer, regioisomer or stereoisomer may have a higherlevel of activity than another isomer, tautomer, regioisomer orstereoisomer.

Compounds designed, selected and/or optimized by methods describedabove, once produced, can be characterized using a variety of assaysknown to those skilled in the art to determine whether the compoundshave biological activity. For example, the molecules can becharacterized by conventional assays, including but not limited to thoseassays described below, to determine whether they have a predictedactivity, binding activity and/or binding specificity.

Furthermore, high-throughput screening can be used to speed up analysisusing such assays. As a result, it can be possible to rapidly screen themolecules described herein for activity, using techniques known in theart. General methodologies for performing high-throughput screening aredescribed, for example, in Devlin (1998) High Throughput Screening,Marcel Dekker; and U.S. Pat. No. 5,763,263. High-throughput assays canuse one or more different assay techniques including, but not limitedto, those described below.

All publications and patent documents cited herein are incorporatedherein by reference as if each such publication or document wasspecifically and individually indicated to be incorporated herein byreference. Citation of publications and patent documents is not intendedas an admission that any is pertinent prior art, nor does it constituteany admission as to the contents or date of the same. The inventionhaving now been described by way of written description, those of skillin the art will recognize that the invention can be practiced in avariety of embodiments and that the foregoing description and examplesbelow are for purposes of illustration and not limitation of the claimsthat follow.

EXAMPLES Example 1 Syntheses of Compounds of the Invention GeneralExperimental NMR

¹H-NMR spectra were taken using CDCl₃ unless otherwise stated and wererecorded at 400 or 500 MHz using a Varian or Oxford instruments magnet(500 MHz) instruments. Multiplicities indicated are s=singlet,d=doublet, t=triplet, q=quartet, quint=quintet, sxt=sextet, m=multiplet,dd=doublet of doublets, dt=doublet of triplets; br indicates a broadsignal.

LCMS and HPLC

Mass: Waters Acquity Ultra Performance LC. HPLC: Products were analyzedby Shimadzu SPD-20A with 150×4.5 mm YMC ODS-M80 column or 150×4.6 mmYMC-Pack Pro C18 column at 1.0 ml/min. Mobile phase was MeCN:H₂O=3:2(containing 0.3% SDS and 0.05% H₃PO₄). Products were purified by HPLC/MS(MeOH—H₂O containing 0.1% ammonium hydroxide) using WatersAutoPurification System with 3100 Mass Detector.

3-(Aminomethyl)-4,6-dimethyl-1,2-dihydropyridin-2-one HCl Salt

To a solution of 2-cyanoacetamide (8.40 g, 100 mmol) and acetylacetone(10.0 g, 100 mmol) in H₂O (200 mL) was added K₂CO₃ (4.00 g, 28.9 mmol).The mixture was stirred at RT for 22 hours. Then the precipitated solidwas filtered with Buchner funnel, washed with ice cold H₂O, and driedunder vacuum pressure to give4,6-dimethyl-2-oxo-1,2-dihydropyridine-3-carbonitrile (13.5 g, 91%yield).

To a solution of 4,6-dimethyl-2-oxo-1,2-dihydropyridine-3-carbonitrile(10.0 g, 67.5 mmol) in MeOH (1.50 L) and conc. HCl (30 mL) was added 10%Pd(OH)₂ (19 g) under N₂ atmosphere. The N₂ gas was displaced by H₂ gasand the mixture was stirred for 26 hours at RT under hydrogenatmosphere. The H₂ gas was displaced by N₂ gas. The mixture was filteredthrough Celite, washed with MeOH and concentrated. The residue wastriturated with EtOH, collected with Buchner funnel, and dried undervacuum pressure to give the titled compound as a white solid (11.5 g,90%). ¹H NMR (400 MHz, DMSO-d₆): δ ppm 11.86 (brs, 1H), 5.98 (s, 1H),3.78 (m, 2H), 2.20 (s, 3H), 2.16 (s, 3H).

5-Bromo-2-methyl-3-nitrobenzoic acid

To a stirred solution of 2-methyl-3-nitrobenzoic acid (5.00 g, 27.6mmol) in H₂SO₄ (20 mL) was added 1,3-dibromo-5,5-dimethylhydantoin (4.34g, 15.20 mmol) at 0° C. The reaction mixture was stirred at 0° C. for 5hours. The reaction mixture was poured onto ice cold water, theresultant precipitated solid was collected, washed with water and driedin vacuo to give the titled compound as a white solid (7.28 g,quantitative yield). ¹H-NMR (400 MHz, DMSO-d₆) δ ppm; 8.31 (s, 1H), 8.17(s, 1H), 2.43 (s, 3H).

Methyl 5-bromo-2-methyl-3-nitrobenzoate

To a stirred solution of 5-bromo-2-methyl-3-nitrobenzoic acid (7.28 g,28.0 mmol) in DMF (100 mL) was added sodium carbonate (11.9 g, 112 mmol)and methyl iodide (15.9 g, 112 mmol). The reaction mixture was stirredat 60° C. for 8 hours. After completion of the reaction, the reactionmixture was filtered and washed with ethyl acetate. The combinedfiltrate was washed with water and the aqueous phase was re-extractedwith ethyl acetate. The combined organic layers were dried overanhydrous sodium sulfate, filtered and concentrated under reducedpressure to afford the titled compound as a solid. (7.74 g, quantitativeyield). ¹H-NMR (400 MHz, CDCl₃) δ (ppm); 8.17 (s, 1H), 7.91 (s, 1H),3.96 (s, 3H), 2.59 (s, 3H).

Methyl 3-amino-5-bromo-2-methylbenzoate

To a stirred solution of methyl 5-bromo-2-methyl-3-nitrobenzoate (7.60g, 27.7 mmol) in aq. EtOH (100 mL of EtOH and 20 mL of H₂O) was addedammonium chloride (4.45 g, 83.1 mmol) and iron (4.64 g, 83.1 mmol). Thereaction mixture was stirred at 80° C. for 5 hours. Then the mixture wasfiltered through Celite and the Celite bed was washed with ethylacetate. The combined filtrate was concentrated in vacuo. The resultantresidue was dissolved in ethyl acetate and water. The aqueous layer wasextracted with ethyl acetate (twice). The combined organic layer driedover anhydrous sodium sulfate, filtered and concentrated under reducedpressure to afford the titled compound as a brown oil (6.67 g, 99%).¹H-NMR (400 MHz, CDCl₃) δ ppm; 7.37 (s, 1H), 6.92 (s, 1H), 3.94 (s, 3H),3.80 (brs, 2H), 2.31 (s, 3H).

Step 1: Synthesis of methyl5-(((trans)-4-((tert-butoxycarbonyl)amino)cyclohexyl)(ethyl)amino)-4′-hydroxy-4-methyl-[1,1′-biphenyl]-3-carboxylate

To a stirred solution of methyl5-bromo-3-(((trans)-4-((tert-butoxycarbonyl)amino)cyclohexyl)(ethyl)amino)-2-methylbenzoate(10 g, 21.3 mmol, see, e.g., WO2012142504 (Attorney Docket No.41478-507001WO)) and (4-hydroxyphenyl)boronic acid (3.5 g, 25.3 mmol) ina mixture of dioxane (225 mL) and water (75 mL), Na₂CO₃ (8.01 g, 75.5mmol) was added and the solution was purged with argon for 30 min. ThenPd(PPh₃)₄ (2.4 g, 2.07 mmol) was added and argon was purged again foranother 15 min. Reaction mass was heated at 100° C. for 4 h. Oncompletion, reaction mixture was diluted with water and extracted withethyl acetate. Combined organic layer was dried over sodium sulfate.Removal of the solvent under reduced pressure followed by columnchromatographic purification afforded the title compound (8.9 g, 87%yield).

Step 2: Synthesis of methyl5-(((trans)-4-((tert-butoxycarbonyl)amino)cyclohexyl)(ethyl)amino)-4′-(2-methoxyethoxy)-4-methyl-[1,1′-biphenyl]-3-carboxylate

To a stirred solution of methyl5-(((trans)-4-((tert-butoxycarbonyl)amino)cyclohexyl)(ethyl)amino)-4′-hydroxy-4-methyl-[1,1′-biphenyl]-3-carboxylate(0.6 g, 1.24 mmol) and 1-bromo-2-methoxyethane (0.519 g, 3.73 mmol) inacetonitrile (6 mL), Cs₂CO₃ (0.485 g, 1.49 mmol) was added and reactionwas stirred at 80° C. for 12 h. On completion, water was added to it andextracted with ethyl acetate. The combined organic layers were driedover anhydrous sodium sulfate and concentrated under reduced pressure.The crude compound was purified by column chromatography to afford thetitle compound (0.6 g, 76.5% yield).

Step 3: Synthesis of tert-butyl((trans)-4-((5-(((4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl) methyl)carbamoyl)-4′-(2-methoxyethoxy)-4-methyl-[1,1′-biphenyl]-3-yl(ethyl)-amino)-cyclohexyl)carbamate

Aqueous NaOH (0.066 g, 1.66 mmol in 5 mL H2O) was added to a solution ofmethyl5-(((trans)-4-((tert-butoxycarbonyl)amino)cyclohexyl)(ethyl)amino)-4′-(2-methoxyethoxy)-4-methyl-[1,1′-biphenyl]-3-carboxylate(0.6 g, 1.11 mmol) in EtOH (10 mL) and stirred at 60° C. for 1 h. Aftercompletion of the reaction, ethanol was removed under reduced pressureand the residue was acidified using citric acid using to pH 4 wasadjusted using citric acid. Extraction was carried out using 10%methanol in DCM. Combined organic layers were dried, concentrated givingrespective acid (0.5 g, 85.6% yield).

The above acid (0.5 g, 0.95 mmol) was then dissolved in DMSO (5 mL) and3-(aminomethyl)-4,6-dimethylpyridin-2(1H)-one (0.288 g, 1.90 mmol) andtriethyl amine (0.096 g, 0.950 mmol) was added to it. The reactionmixture was stirred at room temperature for 15 min before PyBop (0.741g, 1.42 mmol) was added to it and stirring was continued for overnightat room temperature. After completion of the reaction, reaction mass waspoured into ice and extraction was carried out using 10% MeOH/DCM.Combined organic layers were dried over sodium sulfate and concentratedunder reduced pressure to obtain crude material which then purified bycolumn chromatography to afford the title compound (0.45 g, 71.8%yield).

Step 4: Synthesis ofN-((4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-5-(((trans)-4-(dimethylamino)-cyclohexyl)(ethyl)-amino)-4′-(2-methoxyethoxy)-4-methyl-[1,1′-biphenyl]-3-carboxamide

To a stirred solution oftert-butyl((trans)-4-(5-(((4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)carbamoyl)-4′-(2-methoxyethoxy)-4-methyl-[1,1′-biphenyl]-3-yl)(ethyl)amino)cyclohexyl)carbamate(0.45 g, 0.681 mmol) in DCM (5 mL) at 0° C., TFA (1 mL) was added andreaction was stirred for 2 h at room temperature. After completion,reaction was concentrated to dryness. The residue was then basified withNa₂CO₃ (aq.) to pH 8 and the aqueous layer extracted with 20% methanolin DCM. The combined organic layers were dried over Na₂SO₄ and thesolvent removed under reduced pressure to give Boc-deprotected compound(0.3 g, 78.7% yield).

To a stirred solution of Boc-deprotected compound (0.3 g, 0.535 mmol) indichloromethane (3 mL) was added formaldehyde solution (35-41% aq.)(0.056 g, 1.87 mmol) at 0° C. and stirred for 20 min. Then, NaBH(OAc)₃(0.28 g, 1.33 mmol) was added and stirred for 2 h at 0° C. On completionof the reaction, water was added and extracted with 20% methanol in DCM.The combined organic layers were dried over Na₂SO₄ and the solvent wasremoved under reduced pressure. The crude compound was purified by prep.HPLC to afford the title compound (0.1 g, 31.7% yield).

LCMS: 589.75 (M+1)⁺; TFA-salt: ¹H NMR (DMSO-d₆, 400 MHz) δ 11.47 (brs,1H), 9.48 (brs, 1H), 8.21 (brs, 1H), 7.57 (d, 2H, J=8.0 Hz), 7.40 (s,1H), 7.23 (s, 1H), 7.03 (d, 2H, J=8.8 Hz), 5.87 (s, 1H), 4.29 (d, 2H,J=4.4 Hz), 4.14-4.12 (m, 2H), 3.69-3.66 (m, 2H), 3.32 (s, 3H), 3.13 (m,4H), 2.69-2.68 (m, 6H), 2.24 (s, 3H), 2.21 (s, 3H), 2.11 (s, 3H), 1.96(m, 4H), 1.44 (m, 4H), 0.85 (t, 3H, J=6.8 Hz).

Step 1: Synthesis of methyl5-bromo-3-(((trans)-4-((tert-butoxycarbonyl)-(methyl)-amino)cyclohexyl)(ethyl)amino)-2-methylbenzoate

To a stirred solution of methyl5-bromo-3-(((trans)-4-((tert-butoxycarbonyl)amino)cyclohexyl)(ethyl)amino)-2-methylbenzoate(3 g, 6.41 mmol, see, e.g., WO2012142504) in THF (30 mL), NaH (0.184 g,7.69 mmol) was added at 0° C. and stirred it at same temperature for 20min. Then methyl iodide (9.10 g, 64.10 mmol) was added at 0° C. andreaction was stirred for overnight at room temperature. On completion,reaction was quenched with ice water and extracted with dichloromethane.The combined organic layers were washed with water, dried, concentratedunder reduced pressure. The crude compound was purified by columnchromatography to afford the crude title compound that was used withoutfurther purification (3 g, 97.4% yield).

Step 2: Synthesis of methyl3-(((trans)-4-((tert-butoxycarbonyl)-(methyl)amino)cyclohexyl)(ethyl)amino)-5-(3-hydroxyprop-1-yn-1-yl)-2-methylbenzoate

To a stirred solution of methyl5-bromo-3-(((trans)-4-((tert-butoxycarbonyl)(methyl)amino)cyclohexyl)(ethyl)amino)-2-methylbenzoate(2 g, 4.14 mmol) in dry toluene was added CuI (0.015 g, 0.079 mmol),PPh₃ (0.043 g, 0.165 mmol), PdCl₂(PPh₃)₂ (0.058 g, 0.082 mmol),N,N-diisopropyl amine (1.08 g, 10.78 mmol) and reaction was purged withargon for 15 min. prop-2-yn-1-ol (0.46 g, 8.29 mmol) was added to itreaction was heated at 80° C. at sealed condition for 5 h. Oncompletion, it was quenched with water and extracted with ethyl acetate.Organic layer was dried over Na₂SO₄. The crude compound was purified bycolumn chromatography to afford the title compound (1.2 g, 63.2% yield).

Step 3: Synthesis of methyl5-(3-bromoprop-1-yn-1-yl)-3-(((trans)-4-((tert-butoxycarbonyl)(methyl)amino)cyclohexyl)(ethyl)amino)-2-methylbenzoate

To a stirred solution of methyl3-(((trans)-4-((tert-butoxycarbonyl)(methyl)amino)cyclohexyl)(ethyl)amino)-5-(3-hydroxyprop-1-yn-1-yl)-2-methylbenzoate(1.2 g, 2.62 mmol) in DCM (15 mL), PPh₃ (1.37 g, 5.22 mmol) and CBr₄(1.7 g, 5.10 mmol) were added at 0° C. and reaction was stirred for 4 hat room temperature. On completion, reaction was quenched with ice waterand extracted with dichloromethane. The combined organic layers werewashed with water, dried, concentrated under reduced pressure. The crudematerial was purified by column chromatography to afford the titlecompound (0.5 g, 38.5% yield).

Step 4: Synthesis of methyl3-(((trans)-4-((tert-butoxycarbonyl)(methyl)amino)cyclohexyl)(ethyl)amino)-2-methyl-5-(3-morpholinoprop-1-yn-1-yl)benzoate

To a stirred solution of methyl5-(3-bromoprop-1-yn-1-yl)-3-(((trans)-4-((tert-butoxycarbonyl)-(methyl)amino)cyclohexyl)(ethyl)amino)-2-methylbenzoate (1equiv.) in DMF, morpholine (5 equiv.) was added and reaction was stirredfor 12 h at room temperature. On completion, the reaction was quenchedwith ice water and extracted with dichloromethane. The combined organiclayers were washed with water, dried, concentrated under reducedpressure to afford desired crude title compound that was used in thenext step without further purification (98.7% yield)

Step 5: Synthesis of tert-butyl((trans)-4-(3-(((4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)carbamoyl)-2-methyl-5-(3-morpholinoprop-1-yn-1-yl)phenyl)(ethyl)amino)cyclohexyl)(methyl)carbamate

NaOH (1.5 eq.) was added to a solution of methyl3-(((trans)-4-((tert-butoxycarbonyl)(methyl)amino)cyclohexyl)(ethyl)amino)-2-methyl-5-(3-morpholinoprop-1-yn-1-yl)benzoate(1 equiv.) in EtOH: H₂O (9:1) and stirred at 60° C. for 1 h. Aftercompletion of the reaction, ethanol was removed under reduced pressureand acidified using dilute HCl up to pH 6 and pH 4 was adjusted usingcitric acid. Extraction was carried out using 10% methanol in DCM.Combined organic layers were dried concentrated giving respective acid.

The above acid (1 equiv.) was then dissolved in DMSO and3-(aminomethyl)-4,6-dimethylpyridin-2(1H)-one (2 equiv.) and triethylamine (1 equiv.) was added to it. The reaction mixture was stirred atroom temperature for 15 min before PyBop (1.5 equiv.) was added to itand stirring was continued for overnight at room temperature. Aftercompletion of the reaction, the reaction mass was poured into ice andextraction was carried out using 10% MeOH/DCM. The combined organiclayers were dried over Na₂SO₄ and concentrated under reduced pressure toobtain crude material which then purified first by water followed byacetonitrile washing to afford desired title compound (69.4% yield).

Step 6: Synthesis ofN-((4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-3-(ethyl((trans)-4-(methylamino)cyclohexyl)amino)-2-methyl-5-(3-morpholinoprop-1-yn-1-yl)benzamide

To a stirred solution oftert-butyl((trans)-4-((3-(((4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)carbamoyl)-2-methyl-5-(3-morpholinoprop-1-yn-1-yl)phenyl)(ethyl)amino)cyclohexyl)(methyl)carbamate(1 equiv.) in DCM at 0° C., TFA (3 equiv.) was added and reaction wasstirred for 2 h at room temperature. After completion, reaction wasconcentrated to dryness. The residue was then basified with Na₂CO₃ (aq.)to pH 8 and the aqueous layer was extracted with 20% methanol in DCM.The combined organic layers were dried over Na₂SO₄ and solvent wasremoved under reduced pressure to afford the title compound (99% yield)which was used in the next reaction without further purification.

Step 7: Synthesis ofN-((4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-3-(ethyl((trans)-4-((2-methoxyethyl)(methyl)amino)cyclohexyl)amino)-2-methyl-5-(3-morpholinoprop-1-yn-1-yl)benzamide

To a stirred solution ofN-((4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-3-(ethyl((trans)-4-(methylamino)cyclohexyl)amino)-2-methyl-5-(3-morpholinoprop-1-yn-1-yl)benzamide(1 equiv.) in dichloroethane, 2-methoxyacetaldehyde (10 equiv.) andacetic acid (6 equiv.) was added at 0° C. and stirred for 20 min. ThenNaBH(OAc)₃ (3 equiv.) was added and stirred for 2 h at 0° C. Oncompletion of reaction, water was added and extracted with 20% methanolin DCM. Combined organic layers were dried over Na₂SO₄ and solventremoved under reduced pressure. The crude compound was purified by prep.HPLC to afford target molecule (0.1 g, 33.6% yield).

LCMS: 606.65 (M+1)⁺; TFA salt: ¹H NMR (DMSO-d₆, 400 MHz) δ 11.50 (brs,1H), 9.22 (brs, 1H), 8.18 (t, 1H), 7.24 (s, 1H), 7.09 (s, 1H), 5.86 (s,1H), 4.26-4.25 (m, 4H), 3.66-3.59 (m, 4H), 3.48-3.36 (m, 3H), 3.29-3.17(m, 7H), 3.04-3.01 (m, 3H), 2.69-2.68 (m, 4H), 2.20 (s, 3H), 2.19 (s,3H), 2.11 (s, 3H), 2.00-1.92 (m, 2H), 1.82-1.73 (m, 3H), 1.46 (m, 4H),0.78 (t, 3H, J=6.4 Hz).

Alternative Synthetic Scheme for Compound 2:

Step A: Synthesis of 4-(prop-2-yn-1-yl)morpholine

To a stirred solution of propargyl bromide (50 g, 420 mmol) in acetone(300 mL), Cs₂CO₃ (136.5 g, 420 mmol) was added at 0° C. Then morpholine(36.60 g, 420 mmol) in acetone (200 mL) was added dropwise and reactionwas stirred at room temperature for 16 h. On completion, the reactionmass was filtered and the filtrate was concentrated under reducedpressure to afford the title compound (50 g, crude). The isolatedcompound was used directly in the subsequent coupling step withoutfurther purification.

Step 1: Synthesis of methyl5-bromo-3-(ethyl((trans)-4-(methylamino)cyclohexyl)amino)-2-methylbenzoate

To a stirred solution of methyl5-bromo-3-(((trans)-4-((tert-butoxycarbonyl)(methyl)amino)cyclohexyl)(ethyl)amino)-2-methylbenzoate(30 g, 62.24 mmol) in methanol (100 mL) at 0° C., methanolic HCl (500mL) was added and reaction was stirred for 2 h at room temperature.After completion, reaction was concentrated to dryness. The residue wasbasified with Na₂CO₃ (aq.) to pH 8 and aqueous layer was extracted with10% methanol in DCM (200 mL×3). Combined organic layers were dried overNa₂SO₄ and solvent removed under reduced pressure to afford the titlecompound as colorless oil (25 g, crude). The isolated compound was usedin the next step without further purification.

Step 2: Synthesis of methyl5-bromo-3-(ethyl((trans)-4-((2-methoxyethyl)(methyl)amino)cyclohexyl)amino)-2-methylbenzoate

To a stirred solution of crude methyl5-bromo-3-(ethyl((trans)-4-(methylamino)cyclohexyl)amino)-2-methylbenzoate(25 g, 65.44 mmol), 1-bromo-2-methoxyethane (18.19 g, 130.8 mmol) inacetonitrile (250 mL), K₂CO₃ (18.06 g, 130.8 mmol) and KI (6.51 g, 39.21mmol) were added. The resulting reaction mass was stirred at 65° C. for16 h. On completion, reaction mixture was diluted with water (300 mL)and extracted with DCM (500 mL×3). The combined organic layers werewashed with water, dried over Na₂SO₄ and concentrated under reducedpressure. The crude compound was purified by silica gel columnchromatography to afford the title compound (20 g, 69.3% yield).

¹H NMR (DMSO-d₆, 400 MHz) δ 7.55 (s, 1H), 7.45 (s, 1H), 3.82 (s, 3H),3.32 (m, 4H), 3.20 (s, 3H), 3.05 (q, 2H), 2.61 (m, 1H), 2.32 (s, 3H),2.30 (m, 1H), 2.15 (s, 3H), 1.77-1.67 (m, 4H), 1.37-1.31 (m, 2H),1.24-1.18 (m, 2H), 0.78 (t, 3H, J=6.8 Hz).

Step 3: Synthesis of methyl3-(ethyl((trans)-4-(2-methoxyethyl)(methyl)amino)cyclohexyl)amino)-2-methyl-5-(3-morpholinoprop-1-yn-1-yl)benzoate

To a solution of methyl5-bromo-3-(ethyl((trans)-4-((2-methoxyethyl)(methyl)amino)cyclohexyl)amino)-2-methylbenzoate(30 g, 68.02 mmol), 4-(prop-2-yn-1-yl) morpholine (25.51 g, 204 mmol)and triethylamine (20.61 g, 204 mmol) in DMF (300 mL) was bubbled Argonfor 20 min. Then CuI (3.87 g, 20.36 mmol) and Pd (PPh₃)₄ (7.85 g, 6.79mmol) were added and Argon was bubbled through for further 20 min. Thereaction mixture was heated at 105° C. for 4 h and then cooled to roomtemperature. The reaction was quenched with water (100 mL) and theaqueous phase was extracted with 10% MeOH/DCM (400 mL×3). The combinedorganic extracts were dried over Na₂SO₄, filtered and concentrated. Theresidue was, purified by silica gel column chromatography to afford thetitle compound (21 g, 63.7% yield).

¹H NMR (DMSO-d₆, 400 MHz) δ 7.46 (s, 1H), 7.32 (s, 1H), 3.82 (s, 3H),3.62-3.57 (m, 6H), 3.50 (s, 2H), 3.35-3.32 (m, 2H), 3.21 (s, 3H), 3.17(m, 1H), 3.05 (q, 2H), 2.61-2.58 (m, 2H), 2.38 (s, 3H), 2.33 (m, 1H),2.18 (m, 2H), 1.77-1.70 (m, 4H), 1.36-1.20 (m, 4H), 0.77 (t, 3H, J=6.8Hz), 3H merged in solvent peak.

Step 4: Synthesis ofN-((4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-3-(ethyl((trans)-4-((2-methoxyethyl)(methyl)amino)cyclohexyl)amino)-2-methyl-5-(3-morpholinoprop-1-yn-1-yl)benzamide

Aqueous NaOH (2.59 g, 64.91 mmol in 10 mL H₂O) was added to a solutionof methyl3-(ethyl((trans)-4-((2-methoxyethyl)(methyl)amino)cyclohexyl)amino)-2-methyl-5-(3-morpholinoprop-1-yn-1-yl)benzoate(21 g, 43.29 mmol) in EtOH (100 mL) and stirred at 60° C. for 1 h. Aftercompletion of the reaction, ethanol was removed under reduced pressureand the residue was acidified using dilute HCl up to pH 4 using citricacid. Extraction was carried out using 10° A) MeOH/DCM (200 mL×3).Combined organic layers were dried concentrated giving respective acid(15.5 g, 76% yield).

To the solution of above acid (15.5 g, 32.90 mmol) in DMSO (50 mL),3-(aminomethyl)-4,6-dimethylpyridin-2(1H)-one (10 g, 65.80 mmol) andtriethyl amine (23 mL, 164.5 mmol) were added. The reaction mixture wasstirred at room temperature for 15 min before PyBop (25.66 g, 49.34mmol) was added to it at 0° C. and further stirred for overnight at roomtemperature. After completion, the reaction mass was poured into icewater (100 mL) and extraction was carried out using 10% MeOH/DCM (200mL×3). Combined organic layers were dried over Na₂SO₄ and concentratedunder reduced pressure. The crude compound was purified by columnchromatography over basic alumina eluting with MeOH:DCM to afford thetitle compound (11 g, 55.3% yield).

LCMS: 606.50 (M+1)⁺; ¹H NMR (MeOD, 400 MHz) δ 7.23 (s, 1H), 7.09 (s,1H), 6.11 (s, 1H), 4.46 (s, 2H), 3.74-3.72 (m, 4H), 3.51 (s, 2H), 3.47(t, 2H, J=5.6 Hz), 3.32 (s, 3H), 3.07 (q, 2H, J=7.2 Hz), 2.64-2.63 (m,7H), 2.38 (m, 1H), 2.37 (s, 3H), 2.27 (s, 3H), 2.26 (s, 3H), 2.25 (s,3H), 1.89-1.86 (m, 4H), 1.50-1.30 (m, 4H), 0.83 (t, 3H, J=7.2 Hz).

Compound 105 Methyl 5-bromo-2-methyl-3-[(oxan-4-yl)amino]benzoate

To a stirred solution of methyl 3-amino-5-bromo-2-methylbenzoate (40.2g, 165 mmol) in CH₂Cl₂ (500 mL) and AcOH (60 mL) was addeddihydro-2H-pyran-4-one (17.3 g, 173 mmol) and sodiumtriacetoxyborohydride (73.6 g, 330 mmol). The reaction mixture wasstirred at RT for 20 hours. Then saturated NaHCO₃ aq. was added and themixture was separated. The aqueous layer was extracted with CH₂Cl₂ andthe combined organic layer was concentrated in vacuo. The residue wastriturated with ethyl ether, and resultant precipitate was collected toafford the titled compound as a white solid (39.1 g, 72%). ¹H-NMR (400MHz, DMSO-d₆) δ ppm; 7.01 (s, 1H), 6.98 (s, 1H), 5.00 (d, J=7.6 Hz, 1H),3.84-3.87 (m, 2H), 3.79 (s, 3H), 3.54-3.56 (m, 1H), 3.43 (m, 2H), 2.14(s, 3H), 1.81-1.84 (m, 2H), 1.47-1.55 (m, 2H).

Methyl 5-bromo-3-[ethyl(oxan-4-yl)amino]-2-methylbenzoate

To a stirred solution of methyl5-bromo-2-methyl-3-[(oxan-4-yl)amino]benzoate (39.1 g, 119 mmol) inCH₂Cl₂ (400 mL) and AcOH (40 mL) was added acetaldehyde (24.7 g, 476mmol) and sodium triacetoxyborohydride (79.6 g, 357 mmol). The reactionmixture was stirred at RT for 24 hours. Then saturated NaHCO₃ aq. wasadded and the mixture was separated. The aqueous layer was extractedwith CH₂Cl₂ and the combined organic layer was concentrated in vacuo.The residue was purified by silica gel column chromatography(SiO₂Heptane/EtOAc=3/1) to give the titled compound as a viscous oil(44.1 g, quantitative yield). ¹H-NMR (400 MHz, DMSO-d₆) δ ppm; 7.62 (s,1H), 7.52 (s, 1H), 3.80 (m, 5H), 3.31 (m, 2H), 2.97-3.05 (m, 2H),2.87-2.96 (m, 1H), 2.38 (s, 3H), 1.52-1.61 (m, 2H), 1.37-1.50 (m, 2H),0.87 (t, J=6.8 Hz, 3H).

tert-Butyl4-((3-(ethyl(tetrahydro-2H-pyran-4-yl)amino)-5-(methoxycarbonyl)-4-methylphenyl)ethynyl)piperidine-1-carboxylate

To a solution of methyl5-bromo-3-(ethyl(tetrahydro-2H-pyran-4-yl)amino)-2-methylbenzoate (1.80g, 5.05 mmol) and tert-butyl 4-ethynylpiperidine-1-carboxylate (1.80 g,8.59 mmol) in DMF (40 ml) was added triethylamine (2.82 ml, 20.2 mmol)and Copper(I) iodide (0.096 g, 0.505 mmol). The reaction mixture wasdegassed by bubbling nitrogen for 15 min. Thentetrakis(triphenylphosphine)palladium(0) (0.292 g, 0.253 mmol) wasintroduced and degassed for additional 10 min by bubbling nitrogen. Thereaction mixture was heated at 80° C. for 6 h. The reaction was quenchedwith sat. NaHCO₃, extracted with TBME (3×40 mL), dried over Na₂SO₄,filtered and concentrated. The residue was purified by chromatography(0% to 40% AcOEt/Heptane) to give the titled compound (2.40 g, 98%yield). ¹H-NMR (500 MHz) 3 ppm; 7.65 (s, 1H), 7.28 (s, 1H), 3.97 (brd,J=11.3 Hz, 2H), 3.90 (s, 3H), 3.76 (m, 2H), 3.34 (dt, J=2.0, 11.7 Hz,2H), 3.24 (ddd, J=3.4, 8.8, 12.2 Hz, 2H), 3.08 (brs, 2H), 2.98 (brs,1H), 2.80 (dddd, J=3.9, 3.9, 3.9, 3.9 Hz, 1H), 2.52 (s, 3H), 1.87 (m,2H), 1.60-1.74 (m, 6H), 1.48 (s, 9H), 0.89 (t, J=6.8 Hz, 3H)); MS (ESI)[M+H]⁺485.4.

5-((1-(tert-Butoxycarbonyl)piperidin-4-yl)ethynyl)-3-(ethyl(tetrahydro-2H-pyran-4-yl)amino)-2-methylbenzoicacid

To a solution of tert-butyl4-((3-(ethyl(tetrahydro-2H-pyran-4-yl)amino)-5-(methoxycarbonyl)-4-methylphenyl)ethynyl)piperidine-1-carboxylate(2.4 g, 4.95 mmol) in ethanol (20.0 mL) was added a solution of sodiumhydroxide (0.565 g, 14.1 mmol) in water (3.0 ml) at rt. The reactionmixture was heated at 60° C. for 6 h. The reaction was quenched with 1MHCl (5 mL) and then excess citric acid solution to adjust to the pH to5. The mixture was concentrated to remove EtOH and the remaining aqueousphase was extracted with AcOEt (2×40 mL). The organic layers werecombined, dried over Na₂SO₄, filtered and concentrated. The residue waspurified by chromatography (10%-100% AcOEt/Heptane) to give the titledcompound (2.30 g, 99% yield). ¹H-NMR (500 MHz) δ ppm; 7.82 (s, 1H), 7.35(s, 1H), 3.98 (brd, J=11.3 Hz, 2H), 3.77 (m, 2H), 3.35 (dt, J=1.5, 11.3Hz, 2H), 3.25 (ddd, J=3.4, 8.3, 12.2 Hz, 2H), 3.11 (brs, 2H), 3.00 (brs,1H), 2.81 (dddd, J=3.9, 3.9, 3.9, 3.9 Hz, 1H), 2.60 (s, 3H), 1.88 (m,2H), 1.60-1.78 (m, 6H), 1.48 (s, 9H), 0.90 (t, J=6.8 Hz, 3H); MS (ESI)[M+H]⁺471.4.

tert-Butyl4-((3-(((4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)carbamoyl)-5-(ethyl(tetrahydro-2H-pyran-4-yl)amino)-4-methylphenyl)ethynyl)piperidine-1-carboxylate

To a solution of5-((1-(tert-butoxycarbonyl)piperidin-4-yl)ethynyl)-3-(ethyl(tetrahydro-2H-pyran-4-yl)amino)-2-methylbenzoicacid (1.06 g, 2.25 mmol) in DMSO (5.8 mL) at rt was added triethylamine(0.90 mL, 6.44 mmol) and(4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methanaminium chloride(0.405 g, 2.15 mmol). The clear solution become heterogenous. Then HOBT(0.493 g, 3.22 mmol) and EDC (0.617 g, 3.22 mmol) were added and theresulting reaction mixture was stirred at rt overnight. The reaction wasquenched with water (80 mL) and the slurry was stirred for 1 h at rt.The slurry was filtrated and the cake was washed with water (2×20 mL).The collected solid was dried under vacuum to give the titled compound(1.27 g, 98% yield). ¹H-NMR (500 MHz, CD₃OD) 8 ppm; 7.22 (s, 1H), 7.08(d, J=1.0 Hz 1H), 6.11 (s, 1H), 4.45 (s, 2H), 3.92 (brd, J=10.8 Hz, 2H),3.78 (dd, J=4.4, 5.4 Hz, 1H), 3.75 (dd, J=4.4, 5.4 Hz, 1H), 3.36 (t,J=11.7 Hz, 2H), 3.21 (br t, J=8.3 Hz, 2H), 3.07 (q, J=7.3 Hz, 2H), 3.01(dddd, J=3.9, 3.9, 11.3, 11.3 Hz, 1H), 2.84 (dddd, J=3.4, 3.4. 3.9, 3.9Hz, 1H), 2.38 (s, 3H), 2.28 (s, 3H), 2.25 (s, 3H), 1.88 (m, 2H), 1.70((brd, J=12.2 Hz, 2H), 1.60 (m, 4H), 1.47 (s, 9H), 0.87 (t, J=7.3 Hz,3H); MS (ESI) [M+H]⁺605.6.

N-((4,6-Dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-3-(ethyl(tetrahydro-2H-pyran-4-yl)amino)-2-methyl-5-(piperidin-4-ylethynyl)benzamide

To a solution of tert-butyl4-((3-(((4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)carbamoyl)-5-(ethyl(tetrahydro-2H-pyran-4-yl)amino)-4-methylphenyl)ethynyl)piperidine-1-carboxylate(250 mg, 0.413 mmol) in DCM (3 mL) was added 4M HCl in 1,4-dioxane (3mL, 12.0 mmol) at 20° C. The mixture was stirred at 20° C. for 1 h. LCMSindicated that the reaction was completed. The reaction mixture wasdirectly concentrated and the residue was dissolved in DCM and thenneutralized with sat. NaHCO₃/brine. The organic layer was dried (Na₂SO₄)and filtered. And the filtrate was concentrated. The residue was usedfor alkylation without further purification (209 mg, 100%). ¹H-NMR (500MHz, CD₃OD) δ ppm 7.21 (brs, 1H), 7.07 (brs, 1H), 6.11 (s, 1H), 4.46 (s,2H), 3.95-3.89 (m, 2H), 3.39-3.34 (m, 2H), 3.08 (q, J=7.0 Hz, 2H),3.06-2.98 (m, 3H), 2.79-2.72 (m, 1H), 2.72-2.65 (m, 2H), 2.38 (s, 3H),2.28 (s, 3H), 2.25 (s, 3H), 1.94-1.88 (m, 2H), 1.73-1.68 (m, 2H),1.68-1.56 (m, 4H), 0.85 (t, J=7.0 Hz, 3H); MS (ESI) [M+H]⁺505.5.

N-((4,6-Dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-3-(ethyl(tetrahydro-2H-pyran-4-yl)amino)-2-methyl-5-((1-methylpiperidin-4-yl)ethynyl)benzamide

To a solution ofN-((4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-3-(ethyl(tetrahydro-2H-pyran-4-yl)amino)-2-methyl-5-(piperidin-4-ylethynyl)benzamide(100 mg, 0.198 mmol) in methanol (5 mL) was added 35% formaldehyde inH₂O (0.155 mL, 1.98 mmol) at 0° C. After stirring at 0° C. for 10 min,sodium cyanoborohydride (24.9 mg, 0.396 mmol) was added. The resultingmixture was stirred at 0° C. for 1 h. LCMS indicated that the reactionwas completed. The reaction was quenched with sat. NaHCO₃/brine andextracted with EtAOc/Heptane. The organic layer was dried (Na₂SO₄),filtered and concentrated. The residue was purified by chromatography(10 g column, MeOH/DCM=1:9, and then MeOH/7 M NH₃ in MeOH/DCM=1:1:8) toafford the titled compound (96.0 mg, 93%). ¹H-NMR (500 MHz, CD₃OD) δ ppm7.22 (brs, 1H), 7.08 (brs, 1H), 6.10 (s, 1H), 4.46 (s, 2H), 3.94-3.87(m, 2H), 3.35-3.30 (m, 2H), 3.07 (q, J=7.0 Hz, 2H), 3.04-2.97 (m, 1H),2.79-2.71 (m, 2H), 2.67-2.58 (m, 1H), 2.38 (s, 3H), 2.28 (s, 3H), 2.28(s, 3H), 2.25 (s, 3H), 2.28-2.21 (m, 2H), 1.97-1.91 (m, 2H), 1.78-1.67(m, 4H), 1.64-1.54 (m, 2H), 0.85 (t, J=7.0 Hz, 3H); MS (ESI)[M+H]⁺519.4.

Example 2 Bioassay protocol and General Methods Protocol for Wild-Typeand Mutant PRC2 Enzyme Assays

General Materials.

S-adenosylmethionine (SAM), S-adenosylhomocyteine (SAH), bicine, KCl,Tween20, dimethylsulfoxide (DMSO) and bovine skin gelatin (BSG) werepurchased from Sigma-Aldrich at the highest level of purity possible.Dithiothreitol (DTT) was purchased from EMD. ³H-SAM was purchased fromAmerican Radiolabeled Chemicals with a specific activity of 80 Ci/mmol.384-well streptavidin Flashplates were purchased from PerkinElmer.

Substrates.

Peptides representative of human histone H3 residues 21-44 containingeither an unmodified lysine 27 (H3K27me0) or dimethylated lysine 27(H3K27me2) were synthesized with a C-terminal G (K-biotin)linker-affinity tag motif and a C-terminal amide cap by 21^(st) CenturyBiochemicals. The peptides were high-performance liquid chromatography(HPLC) purified to greater than 95% purity and confirmed by liquidchromatography mass spectrometry (LC-MS). The sequences are listedbelow.

H3K27me0: (SEQ ID NO: 101) ATKAARKSAPATGGVKKPHRYRPGGK(biotin)-amideH3K27me2: (SEQ ID NO: 102) ATKAARK(me2)SAPATGGVKKPHRYRPGGK(biotin)-amide

Chicken erythrocyte oligonucleosomes were purified from chicken bloodaccording to established procedures.

Recombinant PRC2 Complexes.

Human PRC2 complexes were purified as 4-component enzyme complexesco-expressed in Spodoptera frugiperda (sf9) cells using a baculovirusexpression system. The subunits expressed were wild-type EZH2(NM_(—)004456) or EZH2 Y641F, N, H, S or C mutants generated from thewild-type EZH2 construct, EED (NM_(—)003797), Suz12 (NM_(—)015355) andRbAp48 (NM_(—)005610). The EED subunit contained an N-terminal FLAG tagthat was used to purify the entire 4-component complex from sf9 celllysates. The purity of the complexes met or exceeded 95% as determinedby SDS-PAGE and Agilent Bioanalyzer analysis. Concentrations of enzymestock concentrations (generally 0.3-1.0 mg/mL) was determined using aBradford assay against a bovine serum albumin (BSA) standard.

General Procedure for PRC2 Enzyme Assays on Peptide Substrates.

The assays were all performed in a buffer consisting of 20 mM bicine(pH=7.6), 0.5 mM DTT, 0.005% BSG and 0.002% Tween20, prepared on the dayof use. Compounds in 100% DMSO (1 μL) were spotted into polypropylene384-well V-bottom plates (Greiner) using a Platemate 2×3 outfitted witha 384-channel pipet head (Thermo). DMSO (1 μL) was added to columns 11,12, 23, 24, rows A-H for the maximum signal control, and SAH, a knownproduct and inhibitor of PRC2 (1 μL) was added to columns 11, 12, 23,24, rows I-P for the minimum signal control. A cocktail (40 μL)containing the wild-type PRC2 enzyme and H3K27me0 peptide or any of theY641 mutant enzymes and H3K27me2 peptide was added by Multidrop Combi(Thermo). The compounds were allowed to incubate with PRC2 for 30 min at25° C., then a cocktail (10 μL) containing a mixture of non-radioactiveand ³H-SAM was added to initiate the reaction (final volume=51 μL). Inall cases, the final concentrations were as follows: wild-type or mutantPRC2 enzyme was 4 nM, SAH in the minimum signal control wells was 1 mMand the DMSO concentration was 1%. The final concentrations of the restof the components are indicated in Table 2, below. The assays werestopped by the addition of non-radioactive SAM (10 μL) to a finalconcentration of 600 μM, which dilutes the ³H-SAM to a level where itsincorporation into the peptide substrate is no longer detectable. 50 μlof the reaction in the 384-well polypropylene plate was then transferredto a 384-well Flashplate and the biotinylated peptides were allowed tobind to the streptavidin surface for at least 1 h before being washedthree times with 0.1% Tween20 in a Biotek ELx405 plate washer. Theplates were then read in a PerkinElmer TopCount platereader to measurethe quantity of ³H-labeled peptide bound to the Flashplate surface,measured as disintegrations per minute (dpm) or alternatively, referredto as counts per minute (cpm).

TABLE 2 Final concentrations of components for each assay variationbased upon EZH2 identity (wild-type or Y641 mutant EZH2) PRC2 Enzyme(denoted by EZH2 Peptide Non-radioactive SAM identity) (nM) (nM) ³H-SAM(nM) Wild-type 185 1800 150 Y641F 200 850 150 Y641N 200 850 150 Y641H200 1750 250 Y641S 200 1300 200 Y641C 200 3750 250

General Procedure for Wild-Type PRC2 Enzyme Assay on OligonucleosomeSubstrate.

The assays were performed in a buffer consisting of 20 mM bicine(pH=7.6), 0.5 mM DTT, 0.005% BSG, 100 mM KCl and 0.002% Tween20,prepared on the day of use. Compounds in 100% DMSO (1 μL) were spottedinto polypropylene 384-well V-bottom plates (Greiner) using a Platemate2×3 outfitted with a 384-channel pipet head (Thermo). DMSO (1 μL) wasadded to columns 11, 12, 23, 24, rows A-H for the maximum signalcontrol, and SAH, a known product and inhibitor of PRC2 (1 μL) was addedto columns 11, 12, 23, 24, rows I-P for the minimum signal control. Acocktail (40 μL) containing the wild-type PRC2 enzyme and chickenerythrocyte oligonucleosome was added by Multidrop Combi (Thermo). Thecompounds were allowed to incubate with PRC2 for 30 min at 25° C., thena cocktail (10 μL) containing a mixture of non-radioactive and ³H-SAMwas added to initiate the reaction (final volume=51 μL). The finalconcentrations were as follows: wild-type PRC2 enzyme was 4 nM,non-radioactive SAM was 430 nM, ³H-SAM was 120 nM, chicken erythrocyteolignonucleosome was 120 nM, SAH in the minimum signal control wells was1 mM and the DMSO concentration was 1%. The assay was stopped by theaddition of non-radioactive SAM (10 μL) to a final concentration of 600which dilutes the ³H-SAM to a level where its incorporation into thechicken erythrocyte olignonucleosome substrate is no longer detectable.50 μL of the reaction in the 384-well polypropylene plate was thentransferred to a 384-well Flashplate and the chicken erythrocytenucleosomes were immobilized to the surface of the plate, which was thenwashed three times with 0.1% Tween20 in a Biotek ELx405 plate washer.The plates were then read in a PerkinElmer TopCount platereader tomeasure the quantity of ³H-labeled chicken erythrocyte oligonucleosomebound to the Flashplate surface, measured as disintegrations per minute(dpm) or alternatively, referred to as counts per minute (cpm).

% Inhibition Calculation

${\% \mspace{14mu} {inh}} = {100 - {( \frac{{dpm}_{cmpd} - {dpm}_{\min}}{{dpm}_{\max} - {dpm}_{\min}} ) \times 100}}$

Where dpm=disintegrations per minute, cmpd=signal in assay well, and minand max are the respective minimum and maximum signal controls.

Four-Parameter IC₅₀ Fit

$Y = {{Bottom} + \frac{( {{Top} - {Bottom}} )}{1 + ( \frac{X}{I\; C_{50}} )^{{Hill}\mspace{14mu} {Coefficient}}}}$

Where top and bottom are the normally allowed to float, but may be fixedat 100 or 0 respectively in a 3-parameter fit. The Hill Coefficientnormally allowed to float but may also be fixed at 1 in a 3-parameterfit. Y is the % inhibition and X is the compound concentration.

IC₅₀ values for the PRC2 enzyme assays on peptide substrates (e.g., EZH2wild type and Y641F) are presented in Table 3 below.

WSU-DLCL2 Methylation Assay

WSU-DLCL2 suspension cells were purchased from DSMZ (German Collectionof Microorganisms and Cell Cultures, Braunschweig, Germany).RPMI/Glutamax Medium, Penicillin-Streptomycin, Heat Inactivated FetalBovine Serum, and D-PBS were purchased from Life Technologies, GrandIsland, N.Y., USA. Extraction Buffer and Neutralization Buffer (5×) werepurchased from Active Motif, Carlsbad, Calif., USA. Rabbit anti-HistoneH3 antibody was purchased from Abcam, Cambridge, Mass., USA. Rabbitanti-H3K27me3 and HRP-conjugated anti-rabbit-IgG were purchased fromCell Signaling Technology, Danvers, Mass., USA. TMB “Super Sensitive”substrate was sourced from BioFX Laboratories, Owings Mills, Md., USA.IgG-free Bovine Serum Albumin was purchased from Jackson ImmunoResearch,West Grove, Pa., USA. PBS with Tween (10×PBST) was purchased from KPL,Gaithersburg, Md., USA. Sulfuric Acid was purchased from Ricca Chemical,Arlington, Tex., USA. Immulon ELISA plates were purchased from Thermo,Rochester, N.Y., USA. V-bottom cell culture plates were purchased fromCorning Inc., Corning, N.Y., USA.V-bottom polypropylene plates werepurchased from Greiner Bio-One, Monroe, N.C., USA.

WSU-DLCL2 suspension cells were maintained in growth medium (RPMI 1640supplemented with 10% v/v heat inactivated fetal bovine serum and 100units/mL penicillin-streptomycin) and cultured at 37° C. under 5% CO₂.Under assay conditions, cells were incubated in Assay Medium (RPMI 1640supplemented with 20% v/v heat inactivated fetal bovine serum and 100units/mL penicillin-streptomycin) at 37° C. under 5% CO₂ on a plateshaker.

WSU-DLCL2 cells were seeded in assay medium at a concentration of 50,000cells per mL to a 96-well V-bottom cell culture plate with 200 μL perwell. Compound (1 μL) from 96 well source plates was added directly toV-bottom cell plate. Plates were incubated on a titer-plate shaker at37° C., 5% CO₂ for 96 hours. After four days of incubation, plates werespun at 241×g for five minutes and medium was aspirated gently from eachwell of cell plate without disturbing cell pellet. Pellet wasresuspended in 200 μL DPBS and plates were spun again at 241×g for fiveminutes. The supernatant was aspirated and cold (4° C.) Extractionbuffer (100 μL) was added per well. Plates were incubated at 4° C. onorbital shaker for two hours. Plates were spun at 3427×g×10 minutes.Supernatant (80 μL per well) was transferred to its respective well in96 well V-bottom polypropylene plate. Neutralization Buffer 5× (20 μLper well) was added to V-bottom polypropylene plate containingsupernatant. V-bottom polypropylene plates containing crude histonepreparation (CHP) were incubated on orbital shaker x five minutes. CrudeHistone Preparations were added (2 μL per well) to each respective wellinto duplicate 96 well ELISA plates containing 100 μL Coating Buffer(1×PBS+BSA 0.05% w/v). Plates were sealed and incubated overnight at 4°C. The following day, plates were washed three times with 300 μL perwell 1×PBST. Wells were blocked for two hours with 300 μL per well ELISADiluent ((PBS (1×) BSA (2% w/v) and Tween20 (0.05% v/v)). Plates werewashed three times with 1×PBST. For the Histone H3 detection plate, 100μL per well were added of anti-Histone-H3 antibody (Abeam, ab1791)diluted 1:10,000 in ELISA Diluent. For H3K27 trimethylation detectionplate, 100 μL per well were added of anti-H3K27me3 diluted 1:2000 inELISA diluent. Plates were incubated for 90 minutes at room temperature.Plates were washed three times with 300 μL 1×PBST per well. For HistoneH3 detection, 100 μL of HRP-conjugated anti-rabbit IgG antibody dilutedto 1:6000 in ELISA diluent was added per well. For H3K27me3 detection,100 μL of HRP conjugated anti-rabbit IgG antibody diluted to 1:4000 inELISA diluent was added per well. Plates were incubated at roomtemperature for 90 minutes. Plates were washed four times with 1×PBST300 μL per well. TMB substrate 100 μL was added per well. Histone H3plates were incubated for five minutes at room temperature. H3K27me3plates were incubated for 10 minutes at room temperature. The reactionwas stopped with sulfuric acid 1N (100 μL per well). Absorbance for eachplate was read at 450 nm.

First, the ratio for each well was determined by:

$( \frac{H\; 3K\; 27{me}\; 3\mspace{14mu} {OD}\; 450\mspace{14mu} {value}}{{Histone}\mspace{14mu} H\; 3\mspace{14mu} {OD}\; 450\mspace{14mu} {value}} )$

Each plate included eight control wells of DMSO only treatment (MinimumInhibition) as well as eight control wells for maximum inhibition(Background wells).

The average of the ratio values for each control type was calculated andused to determine the percent inhibition for each test well in theplate. Test compound was serially diluted three-fold in DMSO for a totalof ten test concentrations, beginning at 25 μM. Percent inhibition wasdetermined and IC₅₀ curves were generated using duplicate wells perconcentration of compound. IC₅₀ values for this assay are presented inTable 3 below.

${{Percent}\mspace{14mu} {Inhibition}} = {100 - ( {( \frac{( {{Individual}\mspace{14mu} {Test}\mspace{14mu} {Sample}\mspace{14mu} {Ratio}} ) - ( {{Background}\mspace{14mu} {Avg}\mspace{14mu} {Ratio}} )}{( {{Minimum}\mspace{14mu} {Inhibition}\mspace{14mu} {Ratio}} ) - ( {{Background}\mspace{14mu} {Average}\mspace{14mu} {Ratio}} )} )*100} )}$

Cell Proliferation Analysis

WSU-DLCL2 suspension cells were purchased from DSMZ (German Collectionof Microorganisms and Cell Cultures, Braunschweig, Germany).RPMI/Glutamax Medium, Penicillin-Streptomycin, Heat Inactivated FetalBovine Serum were purchased from Life Technologies, Grand Island, N.Y.,USA. V-bottom polypropylene 384-well plates were purchased from GreinerBio-One, Monroe, N.C., USA. Cell culture 384-well white opaque plateswere purchased from Perkin Elmer, Waltham, Mass., USA. Cell-Titer Glo®was purchased from Promega Corporation, Madison, Wis., USA. SpectraMaxM5 plate reader was purchased from Molecular Devices LLC, Sunnyvale,Calif., USA.

WSU-DLCL2 suspension cells were maintained in growth medium (RPMI 1640supplemented with 10% v/v heat inactivated fetal bovine serum andcultured at 37° C. under 5% CO₂. Under assay conditions, cells wereincubated in Assay Medium (RPMI 1640 supplemented with 20% v/v heatinactivated fetal bovine serum and 100 units/mL penicillin-streptomycin)at 37° C. under 5% CO₂.

For the assessment of the effect of compounds on the proliferation ofthe WSU-DLCL2 cell line, exponentially growing cells were plated in384-well white opaque plates at a density of 1250 cell/ml in a finalvolume of 50 μl of assay medium. A compound source plate was prepared byperforming triplicate nine-point 3-fold serial dilutions in DMSO,beginning at 10 mM (final top concentration of compound in the assay was20 μM and the DMSO was 0.2%). A 100 nL aliquot from the compound stockplate was added to its respective well in the cell plate. The 100%inhibition control consisted of cells treated with 200 nM finalconcentration of staurosporine and the 0% inhibition control consistedof DMSO treated cells. After addition of compounds, assay plates wereincubated for 6 days at 37° C., 5% CO₂, relative humidity >90% for 6days. Cell viability was measured by quantization of ATP present in thecell cultures, adding 35 μl of Cell Titer Glo® reagent to the cellplates. Luminescence was read in the SpectraMax MS. The concentrationinhibiting cell viability by 50% was determined using a 4-parametric fitof the normalized dose response curves. IC₅₀ values for this assay arealso presented in Table 3 below. The mass spectral data for thesecompounds are also listed in Table 3 below.

TABLE 3 WSU WT EZH2 ELISA pro- EZH2 IC₅₀ MS H3K27me3 liferation IC50peptide v2 (free Compound# IC50 (uM) IC50 (uM) (uM) (μM) form) 1 0.0770.0230 <0.005 588.37 2 0.11043 0.38533 0.01498 605.81 105 0.058-0.1500.325 <0.01 0.0084 518.3257

Example 3 Derivation Of the Lowest Cytotoxic Concentration (LCC)

It is well established that cellular proliferation proceeds through celldivision that results in a doubling of the number of cells afterdivision, relative to the number of cells prior to division. Under afixed set of environmental conditions (e.g., pH, ionic strength,temperature, cell density, medium content of proteins and growthfactors, and the like) cells will proliferate by consecutive doubling(i.e., division) according to the following equation, provided thatsufficient nutrients and other required factors are available.

$\begin{matrix}{N_{t} = {N_{0} \times 2^{\frac{t}{t_{D}}}}} & ( {A{.1}} )\end{matrix}$

where N_(t) is the cell number at a time point (t) after initiation ofthe observation period, N₀ is the cell number at the initiation of theobservation period, t is the time after initiation of the observationperiod and t_(D) is the time interval required for cell doubling, alsoreferred to as the doubling time. Equation A.1 can be converted into themore convenient form of an exponential equation in base e, takingadvantage of the equality, 0.693=ln(2).

$\begin{matrix}{N_{t} = {N_{0}^{\frac{0.693\; t}{t_{D}}}}} & ( {A{.2}} )\end{matrix}$

The rate constant for cell proliferation (k_(p)) is inversely related tothe doubling time as follows.

$\begin{matrix}{k_{p} = \frac{0.693}{t_{D}}} & ( {A{.3}} )\end{matrix}$

Combining equation A.2 and A.3 yields,

N _(t) =N ₀ e ^(k) ^(p) ^(t)  (A.4)

Thus, according to equation A.4 cell number is expected to increaseexponentially with time during the early period of cell growth referredto as log-phase growth. Exponential equations like equation A.4 can belinearized by taking the natural logarithm of each side.

ln(N _(t))=ln(N ₀)+k _(p) t  (A.5)

Thus a plot of ln(N_(t)) as a function of time is expected to yield anascending straight line with slope equal to k_(p) and y-intercept equalto ln(N₀).

Changes in environmental conditions can result in a change in the rateof cellular proliferation that is quantifiable as changes in theproliferation rate constant k_(p). Among conditions that may result in achange in proliferation rate is the introduction to the system of anantiproliferative compound at the initiation of the observation period(i.e., at t=0). When an antiproliferative compound has an immediateimpact on cell proliferation, one expects that plots of ln(N_(t)) as afunction of time will continue to be linear at all compoundconcentrations, with diminishing values of k_(p) at increasingconcentrations of compound.

Depending on the mechanistic basis of antiproliferative action, somecompounds may not immediately effect a change in proliferation rate.Instead, there may be a period of latency before the impact of thecompound is realized. In such cases a plot of ln(N_(t)) as a function oftime will appear biphasic, and a time point at which the impact of thecompound begins can be identified as the breakpoint between phases.Regardless of whether a compound's impact on proliferation is immediateor begins after a latency period, the rate constant for proliferation ateach compound concentration is best defined by the slope of theln(N_(t)) vs. time curve from the time point at which compound impactbegins to the end of the observation period of the experiment.

A compound applied to growing cells may affect the observedproliferation in one of two general ways: by inhibiting further celldivision (cytostasis) or by cell killing (cytotoxicity). If a compoundis cytostatic, increasing concentration of compound will reduce thevalue of k_(p) until there is no further cell division. At this point,the rate of cell growth, and therefore the value of k_(p), will be zero.If, on the other hand, the compound is cytotoxic, then the value ofk_(p) will be composed of two rate constants: a rate constant forcontinued cell growth in the presence of the compound (k_(g)) and a rateconstant for cell killing by the compound (k_(d)). The overall rateconstant for proliferation at a fixed concentration of compound willthus be the difference between the absolute values of these opposingrate constants.

k _(p) =|k _(g) |−|k _(d)|  (A.6)

At compound concentrations for which the rate of cell growth exceedsthat of cell killing, the value of k_(p) will have a positive value(i.e., k_(p)>0). At compound concentrations for which the rate of cellgrowth is less than that for cell killing, the value of k_(p) will havea negative value (i.e., k_(p)<0) and the cell number will decrease withtime, indicative of robust cytotoxicity. When k_(g) exactly matchesk_(d) then the overall proliferation rate constant, k_(p), will have avalue of zero. We can thus define the lowest cytotoxic concentration(LCC) as that concentration of compound that results in a value of k_(p)equal to zero, because any concentration greater than this will resultin clearly observable cytotoxicity. Nota bene: at concentrations belowthe LCC there is likely to be cell killing occurring, but at a rate thatis less than that of residual cell proliferation. The treatment here isnot intended to define the biological details of compound action.Rather, the goal here is to merely define a practical parameter withwhich to objectively quantify the concentration of compound at which therate of cell killing exceeds new cell growth. Indeed, the LCC representsa breakpoint or critical concentration above which frank cytotoxicity isobserved, rather than a cytotoxic concentration per se. In this regard,the LCC can be viewed similar to other physical breakpoint metrics, suchas the critical micelle concentration (CMC) used to define theconcentration of lipid, detergent or other surfactant species abovewhich all molecules incorporate into micellar structures.

Traditionally, the impact of antiproliferative compounds on cell growthhas been most commonly quantified by the IC₅₀ value, which is defined asthat concentration of compound that reduces the rate of cellproliferation to one half that observed in the absence of compound(i.e., for the vehicle or solvent control sample). The IC₅₀, however,does not allow the investigator to differentiate between cytostatic andcytotoxic compounds. The LCC, in contrast, readily allows one to makesuch a differentiation and to further quantify the concentration atwhich the transition to robust cytotoxic behavior occurs.

If one limits the observation time window to between the start of impactand the end of the experiment, then the data will generally fit well toa linear equation when plotted as ln(N_(t)) as a function of time (videsupra). From fits of this type, the value of k_(p) can be determined ateach concentration of compound tested. A replot of the value of k_(p) asa function of compound concentration ([I]) will have the form of adescending isotherm, with a maximum value at [I]=0 of k_(max) (definedby the vehicle or solvent control sample) and a minimum value atinfinite compound concentration of k_(min).

$\begin{matrix}{k_{p} = {\frac{( {k_{\max} - k_{\min}} )}{1 + \frac{\lbrack I\rbrack}{I_{mid}}} + k_{\min}}} & ( {A{.7}} )\end{matrix}$

where I_(mid) is the concentration of compound yielding a value of k_(p)that is midway between the values of k_(max) and k_(min) (note that thevalue of I_(mid) is not the same as the IC₅₀, except in the case of acomplete and purely cytostatic compound). Thus, fitting the replot datato equation A.7 provides estimates of k_(max), k_(min) and I_(mid). If acompound is cytostatic (as defined here), the value of k_(min) cannot beless than zero. For cytotoxic compounds, k_(min) will be less than zeroand the absolute value of k_(min) will relate directly to theeffectiveness of the compound in killing cells.

The fitted values derived from equation A.7 can also be used todetermine the value of the LCC. By definition, when [I]=LCC, k_(p)=0.Thus, under these conditions equation A.7 becomes.

$\begin{matrix}{0 = {\frac{( {k_{\max} - k_{\min}} )}{1 + \frac{LCC}{I_{mid}}} + k_{\min}}} & ( {A{.8}} )\end{matrix}$

Algebraic rearrangement of equation A.8 yields an equation for the LCC.

$\begin{matrix}{{LCC} = {I_{mid}\lbrack {( \frac{k_{\max} - k_{\min}}{- k_{\min}} ) - 1} \rbrack}} & ( {A{.9}} )\end{matrix}$

This analysis is simple to implement with nonlinear curve fittingsoftware and may be applied during cellular assays of compound activitythroughout the drug discovery and development process. In this manner,the LCC may provide a valuable metric for the assessment of compound SAR(structure-activity relationship).

Example 4 In Vivo Assays Mice

Female Fox Chase SCID® Mice (CB17/Icr-Prkdc_(scid)/IcrIcoCrl, CharlesRiver Laboratories) or athymic nude mice (Crl:NU(Ncr)-Foxn1_(nu),Charles River Laboratories) were 8 weeks old and had a body-weight (BW)range of 16.0-21.1 g on Day 1 of the study. The animals were fed adlibitum water (reverse osmosis 1 ppm CO and NIH 31 Modified andIrradiated Lab Diet® consisting of 18.0% crude protein, 5.0% crude fat,and 5.0% crude fiber. The mice were housed on irradiated Enrich-o'cobs™bedding in static microisolators on a 12-hour light cycle at 20-22° C.(68-72° F.) and 40-60% humidity. All procedures complied with therecommendations of the Guide for Care and Use of Laboratory Animals withrespect to restraint, husbandry, surgical procedures, feed and fluidregulation, and veterinary care.

Tumor Cell Culture

Human lymphoma cell lines line were obtained from different sources(ATCC, DSMZ), e.g., Karpas-422 obtained from DSMZ. The cell lines weremaintained as suspension cultures in RPMI-1640 medium containing 100units/mL penicillin G sodium salt, 100 g/mL streptomycin, 1% HEPES, and1% L-Glutamine. The medium was supplemented with 20% fetal bovine serum.The cells were cultured in tissue culture flasks in a humidifiedincubator at 37° C., in an atmosphere of 5% CO₂ and 95% air.

In Vivo Tumor Implantation

Human lymphoma cell lines, e.g., Karpas-422 cells, were harvested duringmid-log phase growth, and re-suspended in RPMI-1640 base media and 50%Matrigel™ (BD Biosciences) (RPMI:Matrigel=1:1). Each mouse received1×10⁷ cells (0.2 mL cell suspension) subcutaneously in the right flank.Tumors were calipered in two dimensions to monitor growth as the meanvolume approached the desired 80-120 mm³ range. Tumor size, in mm³, wascalculated from:

${{Tumor}\mspace{14mu} {volume}} = \frac{w^{2} \times l}{2}$

where w=width and l=length, in mm, of the tumor. Tumor weight can beestimated with the assumption that 1 mg is equivalent to 1 mm³ of tumorvolume. After 10-30 days mice with 145-150 mm³ tumors were sorted intotreatment groups with mean tumor volume of 147 mm³.

Test Articles

Test compounds were stored at room temperature and protected from light.On each treatment day, fresh compound formulations were prepared bysuspending the powders in 0.5% sodium carboxymethylcellulose (NaCMC) and0.1% Tween© 80 in deionized water. The vehicle, 0.5% NaCMC and 0.1%Tween® 80 in deionized water, was used to treat the control groups atthe same schedules. Formulations were stored away from light at 4° C.prior to administration. Unless otherwise specified, compounds referredto and tested in this experiment were in their specific salt formsmentioned in this paragraph.

Treatment Plan

Mice were treated at compound doses ranging from 62.5-500 mg/kg on a BID(2 times a day every 12 h) schedule for various amounts of days by oralgavage. Each dose was delivered in a volume of 0.2 mL/20 g mouse (10mL/kg), and adjusted for the last recorded weight of individual animals.The maximal treatment length was 28 days.

Median Tumor Volume (MTV) and Tumor Growth Inhibition (TGI) Analysis

Treatment efficacy was determined on the last treatment day. MTV(n), themedian tumor volume for the number of animals, n, evaluable on the lastday, was determined for each group. Percent tumor growth inhibition (%TGI) can be defined several ways. First, the difference between theMTV(n) of the designated control group and the MTV(n) of thedrug-treated group is expressed as a percentage of the MTV(n) of thecontrol group:

${\% \mspace{14mu} {TGI}} = {( \frac{{{MTV}(n)}_{control} - {{MTV}(n)}_{treated}}{{{MTV}(n)}_{control}} ) \times 100}$

Another way of calculating % TGI is taking the change of the tumor sizefrom day 1 to day n into account with n being the last treatment day.

${\% \mspace{14mu} {TGI}} = {( \frac{{\Delta \; {{MTV}(n)}_{control}} - {\Delta \; {{MTV}(n)}_{treated}}}{\Delta \; {{MTV}(n)}_{control}} ) \times 100}$Δ MTV_(control) = MTV(n)_(control) − MTV(1)_(control)Δ MTV_(treated) = MTV(n)_(treated) − MTV(1)_(treated)

Toxicity

Animals were weighed daily on Days 1-5, and then twice weekly until thecompletion of the study. The mice were examined frequently for overtsigns of any adverse, treatment related side effects, which weredocumented. Acceptable toxicity for the maximum tolerated dose (MTD) wasdefined as a group mean BW loss of less than 20% during the test, andnot more than 10% mortality due to TR deaths. A death is to beclassified as TR if it is attributable to treatment side effects asevidenced by clinical signs and/or necropsy, or due to unknown causesduring the dosing period. A death is to be classified as NTR if there isevidence that the death is unrelated to treatment side effects. NTRdeaths during the dosing interval would typically be categorized as NTRa(due to an accident or human error) or NTRm (due to necropsy-confirmedtumor dissemination by invasion and/or metastasis). Orally treatedanimals that die from unknown causes during the dosing period may beclassified as NTRu when group performance does not support a TRclassification and necropsy, to rule out a dosing error, is notfeasible.

Sampling

On days 7 or 28 during the studies mice were sampled in a pre-specifiedfashion to assess target inhibition in tumors. Tumors were harvestedfrom specified mice under RNAse free conditions and bisected. Frozentumor tissue from each animal was snap frozen in liquid N₂ andpulverized with a mortar and pestle.

Statistical and Graphical Analyses

All statistical and graphical analyses were performed with Prism 3.03(GraphPad) for Windows. To test statistical significance between thecontrol and treated groups over the whole treatment time coursed arepeated measures ANOVA test followed by Dunnets multiple comparisonpost test or a 2 way ANOVA test were employed. Prism reports results asnon-significant (ns) at P>0.05, significant (symbolized by “*”) at0.01<P<0.05, very significant (“**”) at 0.001<P<0.01 and extremelysignificant (“***”) at P<0.001.

Histone Extraction

For isolation of histones, 60-90 mg tumor tissue was homogenized in 1.5ml nuclear extraction buffer (10 mM Tris-HCl, 10 mM MgCl₂, 25 mM KCl, 1%Triton X-100, 8.6% Sucrose, plus a Roche protease inhibitor tablet1836145) and incubated on ice for 5 minutes. Nuclei were collected bycentrifugation at 600 g for 5 minutes at 4° C. and washed once in PBS.Supernatant was removed and histones extracted for one hour, withvortexing every 15 minutes, with 0.4 N cold sulfuric acid. Extracts wereclarified by centrifugation at 10000 g for 10 minutes at 4° C. andtransferred to a fresh microcentrifuge tube containing 10× volume of icecold acetone. Histones were precipitated at −20° C. for 2hours-overnight, pelleted by centrifugation at 10000 g for 10 minutes,and resuspended in water.

ELISA

Histones were prepared in equivalent concentrations in coating buffer(PBS+0.05% BSA) yielding 0.5 ng/uL of sample, and 100 uL of sample orstandard was added in duplicate to 2 96-well ELISA plates (ThermoLabsystems, Immulon 4HBX #3885). The plates were sealed and incubatedovernight at 4° C. The following day, plates were washed 3× with 300uL/well PBST (PBS+0.05% Tween 20; 10×PBST, KPL #51-14-02) on a Bio Tekplate washer. Plates were blocked with 300 uL/well of diluent (PBS+2%BSA+0.05% Tween 20), incubated at RT for 2 hours, and washed 3× withPBST. All antibodies were diluted in diluent. 100 uL/well ofanti-H3K27me3 (CST #9733, 50% glycerol stock 1:1,000) or anti-total H3(Abeam ab1791, 50% glycerol 1:10,000) was added to each plate. Plateswere incubated for 90 min at RT and washed 3× with PBST. 100 uL/well ofanti-Rb-IgG-HRP (Cell Signaling Technology, 7074) was added 1:2,000 tothe H3K27Me3 plate and 1:6,000 to the H3 plate and incubated for 90 minat RT. Plates were washed 4× with PBST. For detection, 100 uL/well ofTMB substrate (BioFx Laboratories, #TMBS) was added and plates incubatedin the dark at RT for 5 min. Reaction was stopped with 100 uL/well 1NH₂SO₄. Absorbance at 450 nm was read on SpectraMax M5 Microplate reader.

7 Day PD Study

In order to test whether a compound can modulate the H3K27me3 histonemark in tumors in vivo, Karpas-422 xenograft tumor bearing mice weretreated with the compound at 62.5, 83.3, 125, 166.7, 250, 333.3, or 500mg/kg BID or vehicle (BID schedule) for 7 days. There were 5 animals pergroup. Animals were euthanized 3 h after the last dose and tumor waspreserved in a frozen state as described above. Following histoneextraction the samples were applied to ELISA assays using antibodiesdirected against the trimethylated state of histone H3K27 (H3K27me3) ortotal histone H3. Based on these data the ratio of globally methylatedto total H3K27 was calculated. The mean global methylation ratios forall groups as measured by ELISA indicate target inhibition rangecompared to vehicle. The design for this experiment is shown in Table4A.

TABLE 4A Dosing Scheme Dose Dosing Dosing Group N Treatment (mg/kg)volume Route Schedule 1 5 Vehicle — 10 μl/g. p.o. BIDx7 2 5 Compound 162.5 10 μl/g p.o. BIDx7 3 5 Compound 1 83.3 10 μl/g p.o. BIDx7 4 5Compound 1 125 10 μl/g p.o. BIDx7 5 5 Compound 1 166.7 10 μl/g p.o.BIDx7 6 5 Compound 1 250 10 μl/g p.o. BIDx7 7 5 Compound 1 333.3 10 μl/gp.o. BIDx7 8 5 Compound 1 500 10 μl/g p.o. BIDx7 9 5 Compound A* 125 10μl/g. p.o. BIDx7 10 5 Compound A 250 10 μl/g. p.o. BIDx7 *Compound A isN-((4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-5-(ethyl(tetrahydro-2H-pyran-4-yl)amino)-4-methyl-4′-(morpholinomethyl)-[1,1′-biphenyl]-3-carboxamide.

Example 5 Efficacy Study with Increasing Doses in Karpas-422 XenograftModel

In order to test whether a compound could induce an anti-tumor effect invivo, Karpas-422 xenograft tumor bearing mice were treated with acompound at, e.g., 62.5, 125, 250, or 500 mg/kg BID for 28 days. Therewere 10 mice per group for the efficacy arm of the experiment. The tumorgrowth over the treatment course of 28 days for vehicle and testcompound treated groups was measured.

Histones were extracted from tumors collected at the end of the study onday 28 for the efficacy cohort (3 h after the last dose for bothcohorts). The H3K27me3 methyl mark was assessed for modulation withtreatment in a dose dependent matter.

The design for this experiment is shown in Table 4B.

TABLE 4B Dosing Scheme Dose Dosing Dosing Group N Treatment (mg/kg)volume Route Schedule 1 10 Vehicle — 10 μl/g. p.o. BIDx28 2 10 Compound1 10 μl/g p.o. BIDx28 3 10 Compound 1 125 10 μl/g. p.o. BIDx28 4 10Compound 1 250 10 μl/g. p.o. BIDx28 5 10 Compound 1 500 10 μl/g. p.o.BIDx28 6 10 Compound A* 250 10 μl/g. p.o. BIDx28 *Compound A isN-((4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-5-(ethyl(tetrahydro-2H-pyran-4-yl)amino)-4-methyl-4′-(morpholinomethyl)-[1,1′-biphenyl]-3-carboxamide.

TV was calculated from caliper measurements by the formula for thevolume of a prolate ellipsoid (L×W²)/2 where L and W are the respectiveorthogonal length and width measurements (mm).

Data were expressed as the mean±standard deviation (SD). The differencesin TV between the vehicle-treated and compound-treated groups wereanalyzed by a repeated measures analysis of variance (ANOVA) followed bythe Dunnett-type multiple comparison test. A value of P<0.05 (two sided)is considered statistically significant. Statistical analyses wereperformed using the Prism 5 software package version 5.04 (GraphPadSoftware, Inc., CA, USA).

The above studies showed that both Compound 1 and Compound A exhibitedtumor stasis and regression in Karpas-422 xenograft model and were welltolerated. See, e.g., FIGS. 1 and 2. Further, the pharmacokinetic andpharmacodynamic properties of Compound 1 or Compound A from the abovestudies are illustrated in FIGS. 3-8. FIG. 3 is a diagram showingconcentration of Compound 1 in tumor at day 7 or day 28 post treatmentor concentration of Compound A in tumor at day 7 post treatment. In thisfigure, “A” though “G” denote 7 days post administration of Compound 1at dosages of 62.5, 83.3, 125, 166.7, 250, 333.3, and 500 mg/kg,respectively; “H” and “I” denote 7 days post administration of CompoundA at dosages of 125 and 250 mg/kg, respectively; and “J” through “L”denote 28 days post administration of Compound 1 at dosages of 62.5, 125and 250 mg/kg, respectively. Note: for samples at day 28, tumors weretoo small for analysis from the group treated with 250 mg/kg Compound Aand the group treated with 500 mg/kg Compound 1; only 1 out of 10 and 5out of 10 were large enough for analysis from groups treated with 250mg/kg Compound 1 and 125 mg/kg Compound 1, respectively. FIG. 4 is adiagram showing concentration of Compound 1 or Compound A in plasma atday 7 or day 28 post treatment. The top dashed line indicates the plasmaprotein binding (PPB) corrected LCC of Compound A and the bottom dashedline indicates PPB corrected LCC of Compound 1. FIG. 5 is a diagramshowing global H3K27me3 methylation in KARPAS-422 tumors from micetreated with Compound 1 or Compound A for 7 days. In this figure, “A”denotes vehicle treatment; “B” though “H” denote treatment with Compound1 at dosages of 62.5, 83.3, 125, 166.7, 250, 333.3, and 500 mg/kg,respectively; and “I” and “J” denote treatment with Compound A atdosages of 125 and 250 mg/kg, respectively. FIG. 6 is a diagram showingglobal H3K27me3 methylation in KARPAS-422 tumors from mice treated withCompound 1 for 28 days. FIG. 7 is a diagram showing global H3K27me3methylation in bone marrow from KARPAS-422 xenograft tumor bearing micetreated with Compound 1 or Compound A for 7 days. In this figure, “A”denotes vehicle treatment; “B” though “H” denote treatment with Compound1 at dosages of 62.5, 83.3, 125, 166.7, 250, 333.3, and 500 mg/kg,respectively; and “I” and “J” denote treatment with Compound A atdosages of 125 and 250 mg/kg, respectively. FIG. 8 is a diagram showingglobal H3K27me3 methylation in bone marrow from KARPAS-422 xenografttumor bearing mice treated with Compound 1 for 28 days. In this figure,“A” denotes vehicle treatment; “B” though “E” denote treatment withCompound 1 at dosages of 62.5, 125, 250, and 500 mg/kg, respectively;and “F” denotes treatment with Compound A at a dosage of 250 mg/kg.

In Examples 6-8 below, experiments and analyses were conducted withmethods and techniques similar to those described in e.g., J. Lin,Pharmaceutical Research, 2006, 23(6):1089-1116; L. Di et al., Comb ChemHigh Throughput Screen. 2008, 11(6):469-76; M. Fonsi et al., Journal ofBiomolecular Screening, 2008, 13:862; E. Sjögren et al., Drug Metab.Dispos. 2012, 40:2273-2279; T. D. Bjornsson, et al., Drug Metab Dispos,2003, 31(7):815-832; J. B. Houston and K. E. Kenworthy, Drug MetabDispos, 2000, 28(3):246-254; and S. W. Grimm et al., Drug Metab

Dispos, 2009, 37(7):1355-1370, the contents of each of which are herebyincorporated by reference in their entireties.

Example 6 Assessment of Metabolic Stability in Liver Microsomes

The metabolic stability of Compounds 1, 2, and 105, were evaluated inliver microsomes from five species, including mice, rats, dogs, monkeys,and humans.

Methods: The incubations were conducted in 96-well plates containing 250μL total volume consisting of 100 mmol/L potassium phosphate buffer (pH7.4), 1 mg/mL liver microsomes, test compound (i.e., Compounds 1, 2, or105) at 8 concentrations, and 2 mg/mL NADPH. The concentrations of thecompounds used for incubation ranged from 45.7 nM to 100 μM. Theaddition of NADPH was used to start the reaction, and the incubationswere done in a shaking water bath at 37° C. for up to 60 minutes. Thereactions were terminated by adding an equal volume of stop solutioncontaining Internal Standard (IS). The samples were then spun in arefrigerated centrifuge at 3000 RPM for a minimum of 5 minutes prior toanalysis. The amount of depletion in the incubation mixes for the testcompound, as determined by using LC/TOFMS, was used to estimateintrinsic clearance values with liver microsomes. The LC/TOFMS systemswere composed of a Shimadzu SIL-HTC autosampler (Kyoto, Japan), twopumps LC-20AD; Shimadzu Corp.), and a column oven (CTO-20AC; ShimadzuCorp.) with a time-of-flight mass spectrometer (AB SCIEX Qstar Elite, ABSciex, Foster City, Calif.). Peak areas of the test compound and IS forassay were integrated by Analyst QS (version 2.0, Applied Biosystems,Foster City, Calif.). Collision activated dissociation (CAD) withnitrogen was used to generate product ions. The optimized instrumentalconditions were under positive ionization mode.

The LC/MS/MS quantification was based on the ratios of peak areas of thetest compound to that of the IS. Peak area calculation and integrationof Compounds 1, 2, or 105 and IS utilized Analyst QS 2.0 (AB Sciex,Foster City, Calif.). Calculations were done using Excel (Office 2010,Microsoft Corp., Redmond, Wash.) and GraphPad Prism v. 5.02 (GraphPadSoftware Inc., La Jolla, Calif.). Data was analyzed and reported basedon appropriate SOPs, such as those described in J. Lin, PharmaceuticalResearch, 2006, 23(6):1089-1116; and Di L et al., Comb Chem HighThroughput Screen. 2008 11(6):469-76.

The depletion of the test compound used for K_(m) and V_(max) valueswith liver microsomes was calculated by plotting against time todetermine the rate of depletion. The rates of depletion were thenplotted using an appropriate kinetic model. The data were calculatedbased on the following equations:

Michaelis-Menten: Cl_(int) (μL/min/mg of liver microsomes)=V_(max)/K_(m)

Hill: Cl_(int) (μL/min/mg of livermicrosomes)=Cl_(max)=V_(max)/K_(s)·(n−1)/(n(n−1)^(1/n))

Cl_(int) (μL/min/g liver)=Cl_(int) (μL/min/mg microsomes)·Scaling Factoror Cl_(int) (μL/min/10⁶ cells)·Scaling Factor.

The results are provided in Table 5 below.

TABLE 5 Estimated Intrinsic Clearance (mL/min/kg) Mouse Rat Dog CynoHuman Compound 1 15.4 14.8 8.1 58.6 10.5 Compound 105 54.7 47.5 64.274.8 42.1 Compound 2 17.6 14.8 34.3 32.8 8.8

All of the three tested compound, i.e., Compounds 1, 2, and 105exhibited low metabolic clearance in human liver microsomes (HLM).Species difference in metabolic clearance was also observed. Cynomolgusmonkeys generally showed higher clearance than other species.

Example 7 Assessment of CYP Induction

The induction of each of Compounds 1, 2, and 105 was evaluated incryopreserved human hepatocytes from a single donor.

Methods: The hepatocytes were obtained from BD Biosciences (Woburn,Mass.), and the appropriate media and DAPI nuclear stain were purchasedfrom Life Technologies (Durham, N.C.). Dulbecco's modified Eagle'sMedium (DMEM), Dulbecco's phosphate buffered saline (DPBS), 100× MEMnon-essential amino acids, 100× penicillin/streptomycin/glutaminesolution, 2× trypan blue were purchased from Mediatech (Manassas, Va.).Fetal bovine serum (FBS) was acquired from Tissue Culture Biologicals(Tulare, Calif.). The 24-well collagen coated plates for mRNA analysiswere acquired from BD Biosciences (San Jose, Calif.). Predesigned probesand primers were used in two triplex assays to assess change in mRNA.The positive controls were β-naphthoflavone for CYP1A (1 and 10μmmol/L), phenobarbital for CYP2B6 (100 μmol/L and 1 mmol/L), andrifampicin for CYP2C9 and CYP3A (1 and 10 μmol/L). DMSO was used as thevehicle (negative) control. CYP form specific assays were performedafter the treatment period, and cells were counted to determineviability.

The cryopreserved hepatocytes were thawed in a 37° C. water bath andplated according to vendor instructions. One tube of hepatocytes wasadded to a 50 mL conical tube containing Life Technologies cryopreservedhepatocyte recovery medium (CHRM). The cells were spun in a Beckmancentrifuge with a GH 3.8 rotor at 800 RPM for 10 minutes. Thesupernatant was removed and the cells were resuspended in plating mediafor counting. After counting, the cells were resuspended at 0.75 millionviable cells/mL. The suspension (0.5 mL/well) was added into a 24-wellcollagen coated plate, or, after the addition of 60 μL of DMEMcontaining 10% FBS, penicillin/streptomycin/glutamine, and MEMnon-essential amino acids, 80 μL of the suspension was added to eachwell of a 96-well collagen coated plate. After swirling and rocking toprovide better plate coverage, the cells were placed in a tissue cultureincubator at 5% CO₂ and 37° C. and allowed to attach overnight. Thecells were then treated using CellzDirect hepatocyte maintenance media.The cells were exposed to Compound 1, 2, or 105 (1 or 10 μmol/L) orvehicle for 48 hours. During the treatment, the maintenance media andtest compounds were replenished every 24 hours.

After completion of the incubation, the cells were washed with DPBS.After washing with DPBS, the cells were fixed using 3.7% p-formaldehydein DPBS for one hour. The formaldehyde was removed and 0.6 μmol/L DAPIin DPBS was added. The cells were stained by DAPI for 20 minutes andthen washed three times with DPBS. Cells were counted using an ArrayScanII (Cellomics, Pittsburgh, Pa.) with a 5× objective lens. Fraction ofhepatocytes remaining was calculated using the number of cells found ata treatment condition divided by cells found with the vehicle control.

After treatment, cells were lysed using buffer RLT from the QiagenRNeasy kit. The mRNA was isolated using the RNeasy kit with DNasetreatment using manufacturer's protocols.

The mRNA concentration was measured via a Nanodrop ND-1000 (Wilmington,Del.). The mRNA concentration was normalized for every sample within adonor. The reverse transcription was performed with a Superscript VILOcDNA synthesis kit from Life Technologies following manufacturer'sdirections. After cDNA synthesis, quantitative real time PCR wasperformed using a 7500 Fast Real Time PCR system from AppliedBiosystems, a wholly owned subsidiary of Life Technologies. The reactioncomponents for real time PCR consisted of: 10 μL of Taqman Fast AdvancedMaster Mix (Life Technologies), 2 μL cDNA, 5 μL nuclease free water(Life Technologies), 1 μL of primer limited FAM labeled assay14500984230 ml for 13-2 microglobulin, 1 μL of primer limited VIClabeled assay HS00167927_m1 for CYP1A2 or HS04183483_g1 for CYP2B6, and1 μL of primer limited NED labeled assay HS04260376_m1 for CYP2C9 orHS00604506_ml for CYP3A4. Calculations of fold difference as compared tovehicle control were done by 7500 Software version 2.0.5 (LifeTechnologies) using the ΔΔC_(t) method. Calculations of E_(max) and EC₅₀were done using GraphPad Prism.

TABLE 6 CYP1A CYP2B6 CYP2C9 CYP3A % % % % % Cell Concentration FoldPositive Fold Positive Fold Positive Fold Positive Remaining Compound(μM) Induction Control Induction Control Induction Control InductionControl vs. Control 1 1 1.04 2.8 0.98 18.0 1.04 43.5 1.04 13.9 107.44 101.14 3.0 1.43 26.3 1.26 52.6 0.99 13.3 99.00 2 1 0.73 NA 0.61 NA 0.57 NA0.59 NA 124.70 10 0.83 NA 0.64 NA 0.73 NA 0.73 NA 120.90 105 1 1.14 3.01.39 25.5 1.20 50.4 1.35 18.1 103.19 10 1.65 4.4 2.84 52.2 1.82 76.32.52 33.8 97.49

As shown in Table 6 above, Compound 1 exhibited no significant induction(except potentially CYP2C9); Compound 2 exhibited no induction (withslightly lower activity); and Compound 105 exhibited induction ofCYP2B6, CYP2C9, and CYP3A. No significant loss of cell viability wasobserved.

Example 8 Assessment of CYP Inhibition

The potential inhibition of enzyme activities of human cytochromes P450(CYP) of Compound 1, 2, or 105 was evaluated using pooled human livermicrosomes.

Methods: The competitive inhibition potential of Compounds 1, 2, and 105was determined by assessing at multiple concentrations on probe CYPreactions near their respective K_(m) values to create IC₅₀ curves inhuman liver microsomes (HLM). The time-dependent inactivation (TDI)potential was also assessed for CYP3A4/5 by evaluating K_(I) andk_(inact) values when appropriate.

A suspension containing PB, HLM, CYP-selective probe substrate, and theinhibitor being tested was added to a 96-well plate. The plates werepreincubated in a 37° C. water bath for approximately 2 minutes. Thereaction was initiated by the addition of NADPH to each well of the96-well plate. The final concentrations for PB, HLM, and NADPH were 100mmol/L (pH 7.4), 0.1 mg/mL, and 2.3 mmol/L, respectively. The CYP probesubstrates and CYP inhibitors used as positive controls and theirrespective concentrations are listed below. The final MeOH concentrationused in each incubation did not exceed 0.8%.

After the appropriate incubation time, an equal volume of the quenchsolution containing IS was added to the appropriate wells. Standard andquality control (QC) samples were prepared using similar components asthe test samples. The blank was prepared with a similar quench solutionas the other samples but did not include IS. The plates were spun for 5minutes in a bench-top centrifuge at 3000 rpm. The samples were analyzedby LC/MS/MS. The incubation condition and the positive controls arelisted in Table 7 below.

TABLE 7 Max. CYP Sub. CYP Inhib. Incub. Internal CYP Probe Conc.Inhibitor Conc. Time Standard Tested Substrate (μmol/L) (PositiveControl) (μmol/L) (min) (LC/MS/MS) CYP1A2 Phenacetin 40 Furafylline 2010 ¹³C₂,¹⁵N-acetaminophen CYP2B6 Bupropion 140 Ticlopidine 5 10²H₆-hydroxybupropion CYP2C8 Amodiaquine 10 Montelukast 12 10²H₃-desethylamodiaquine CYP2C9 Tolbutamide 100 Sulfaphenazole 20 10²H₉-hydroxytolbutamide CYP2C19 (S)-Mephenytoin 30 (S)-Benzylnirvanol 810 ²H₃-4′-hydroxymephenytoin CYP2D6 (±)-Bufuralol 20 Quinidine 2 5²H₉-1′-hydroxybufuralol CYP3A4/5 Midazolam 3 Ketoconazole 1 5¹³C₃-1′-hydroxymidazolam Testosterone 70 (R)-propranolol

For assessing the TDI, the primary incubation, a suspension containingPB, HLM, and the inhibitor stocks was added to a 96-well plate. Theplates were preincubated in a 37° C. water bath for approximately 2minutes. The reaction was initiated by the addition of NADPH to eachwell of the 96-well plate and carried out for 0, 5, 10, 15, 20, and 30minutes. For the no NADPH controls, PB solution was substituted forNADPH stock solution. The final concentrations for PB, HLM, and NADPHwere 100 mmol/L (pH 7.4), 0.2 mg/mL, and 2.3 mmol/L, respectively. Atthe respective times, 12.5 μL of primary incubation suspension wasdiluted 20-fold into pre-incubated secondary incubation mixturecontaining CYP-selective probe substrates in order to assess residualactivity. The probe substrates used was testosterone (250 μmol/L) andthe positive control was troleandomycin.

The HPLC system used was a Shimadzu HPLC system (Kyoto, Japan)consisting of a SIL-HTC autosampler, a DGU-14A degasser, three LC-10ADvppumps, and a CTO-10ACvp column oven. The samples were analyzed on anAPI4000QTrap (AB Sciex, Foster City, Calif.) triple quadrupole massspectrometer using turbo spray ionization under positive ion mode.

Peak area calculation and integration of all monitored metabolites andIS were processed by Analyst 1.6 (AB Sciex, Foster City, Calif.).Quantitation was achieved with the use of calibration curves constructedby plotting the peak area ratio of metabolite to IS againstconcentrations of calibration standards, and was generated by quadraticregression with a 1/x² weighting (y=a x²+b x+c). Percent inhibitioncalculations utilized Excel (Office 2010, Microsoft Corp., Redmond,Wash.), and were plotted using GraphPad Prism (Version 5.02, GraphPadSoftware, Inc., LaJolla, Calif.) based on the assumption of one bindingsite. Nominal concentrations of test compounds and the positive controlswere log transformed in GraphPad Prism. Using the plots of remainingCYP3A4/5 activity versus preincubation time, rate of CYP activity loss(2) was estimated by nonlinear regression. The parameters, k_(inact) andK_(I), were subsequently estimated by fitting to the following equation:

$\lambda = {k_{inact} \times \frac{\lbrack I\rbrack}{K_{I} + \lbrack I\rbrack}}$

where [I] is the initial concentration of Compound 1, 2, or 105.

TABLE 8 Reversible inhibition IC₅₀ (μM) CYP3A4/5 CYP3A4/5 Compound CYP1ACYP2C8 CYP2C9 CYP2C19 CYP2D6 Midazolam Testosterone 1 >100 >100 >10089.57 22.94 40.46 >100 2 >100 >100 >100 >100 >100 >100 >100 105 >10047.43 75.66 33.65 2.95 9.67 >100

TABLE 9 Time-Dependent Inactivation (TDI) assessment Compound K_(I) (μM)k_(inact) (min⁻¹) k_(inact)/K_(I) (μM⁻¹ · min⁻¹) 1 6.4 (3.3**) 0.02770.0043 105 10.2 0.0214 0.0021 2 22.3 0.0376 0.0017

As shown in Table 8 above, Compounds 1 and 2 exhibited no significantreversible inhibition; and Compound 105 exhibited potentiallysignificant inhibition of CYP2D6 and CYP3A4 (midazolam). Also, as shownin Table 9 above, all of Compounds 1, 2, and 105 showed time-,concentration-, and NADPH-dependent inactivation of CYP3A4/5 (usingmidazolam as probe).

The entire disclosure of each of the patent documents and scientificarticles referred to herein is incorporated by reference for allpurposes.

The invention can be embodied in other specific forms without departingfrom the spirit or essential characteristics thereof. The foregoingembodiments are therefore to be considered in all respects illustrativerather than limiting on the invention described herein. The scope of theinvention is thus indicated by the appended claims rather than by theforegoing description, and all changes that come within the meaning andrange of equivalency of the claims are intended to be embraced therein.

What is claimed is:
 1. A compound selected from the group consisting of

and pharmaceutically acceptable salts thereof.
 2. The compound of claim1, wherein the compound is

or a pharmaceutically acceptable salt thereof.
 3. The compound of claim1, wherein the compound is


4. The compound of claim 1, wherein the compound is

or a pharmaceutically acceptable salt thereof.
 5. The compound of claim1, wherein the compound is


6. The compound of claim 1, wherein the compound is

or a pharmaceutically acceptable salt thereof.
 7. The compound of claim1, wherein the compound is


8. A pharmaceutical composition comprising a compound of claim 1 and apharmaceutically acceptable carrier.
 9. A pharmaceutical compositioncomprising a compound of claim 2 and a pharmaceutically acceptablecarrier.
 10. A pharmaceutical composition comprising a compound of claim3 and a pharmaceutically acceptable carrier.
 11. A pharmaceuticalcomposition comprising a compound of claim 4 and a pharmaceuticallyacceptable carrier.
 12. A pharmaceutical composition comprising acompound of claim 5 and a pharmaceutically acceptable carrier.
 13. Apharmaceutical composition comprising a compound of claim 6 and apharmaceutically acceptable carrier.
 14. A pharmaceutical compositioncomprising a compound of claim 7 and a pharmaceutically acceptablecarrier.
 15. A method of treating an EZH2-mediated disorder comprisingadministering to a subject in need thereof a therapeutically effectiveamount of a compound of claim
 1. 16. The method of claim 15, wherein theEZH2-mediated disorder is cancer.
 17. The method of claim 16, whereinthe cancer is lymphoma, leukemia or melanoma.
 18. The method of claim16, wherein the cancer is diffuse large B-cell lymphoma (DLBCL),non-Hodgkin's lymphoma (NHL), follicular lymphoma, chronic myelogenousleukemia (CML), acute myeloid leukemia, acute lymphocytic leukemia,mixed lineage leukemia, or myelodysplastic syndromes (MDS).
 19. Themethod of claim 16, wherein the cancer is malignant rhabdoid tumor. 20.The method of claim 15, wherein the compound is

or a pharmaceutically acceptable salt thereof.